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Silver nanoparticles dispersing in chitosan solution: Preparation by γ-ray irradiation and their antimicrobial activities
Materials Chemistry and Physics 115 (2009) 296–302
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Silver nanoparticles dispersing in chitosan solution: Preparation by ␥-ray irradiation and their antimicrobial activities Rangrong Yoksan a,b,∗ , Suwabun Chirachanchai c a
Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, Thailand Division of Physico-Chemical Processing Technology, Faculty of Agro-Industry, Kasesart University, Bangkok 10900, Thailand c The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand b
a r t i c l e
i n f o
Article history: Received 25 September 2008 Received in revised form 16 November 2008 Accepted 1 December 2008 Keywords: Silver Nanoparticle Chitosan Irradiation Transmission electron microscopy (TEM)
a b s t r a c t Silver nanoparticles were prepared by ␥-ray irradiation–reduction under simple conditions, i.e., air atmosphere, using chitosan as a stabilizer. The nanoparticles were spherical with an average size of 7–30 nm as observed from TEM. The size decreased when chitosan concentration increased, while it increased with increasing ␥-ray dose and initial silver nitrate content. The obtained silver nanoparticles dispersed in a 0.5% (w/v) ␥-ray irradiated chitosan–aqueous acetic acid solution were stable for more than 3 months without tendency to precipitate. The silver nanoparticles exhibited antimicrobial activities against Escherichia coli and Staphylococcus aureus. The results suggest that silver nanoparticles dispersed in chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packaging and biomedical applications. © 2008 Elsevier B.V. All rights reserved.
1. Introduction For many decades, silver nanoparticles have been recognized as an antimicrobial agent [1] and used in many fields including biomedical [2,3], food packaging [4,5], waste water treatment [6], etc. The antimicrobial activities of silver nanoparticles were found to relate to their shapes and sizes [7]. Many attempts have been made to synthesize the silver nanoparticles with controllable shape, size, and size distribution. In most cases, the system for fabricating silver nanoparticles consists of salt precursor, reducing agent, and stabilizer. Silver nitrate (AgNO3 ) is usually applied as the salt precursor. The reduction of silver ions (Ag+ ) to silver particles (Ag0 ) is conventionally achieved by a chemical method using reducing agents such as NaBH4 [8], formamide [9], dimethylformamide [9], triethanolamine [9], hydrazine [10], etc. Although this method is simple and effective, the biological toxicity and the environmental hazard of the residual reducing agent are problems. Recently, reductions by other techniques such as ␥-ray irradiation [11,12], UV irradiation [13], microwave [14], and ultrasonic [15] have been alternative ways to overcome those problems and to
∗ Corresponding author at: Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, Thailand. Tel.: +66 2 562 5097; fax: +66 2 562 5092. E-mail addresses: [email protected], [email protected] (R. Yoksan). 0254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2008.12.001
eliminate the purification step or the removal of reducing agent step. To prevent the aggregation of formed metal nanoparticles, various types of stabilizers are used such as long-chain fatty acids (stearic, palmitic, and lauric acids) [9], lauryl amine, poly(vinyl pyrrolidone) [16], poly(vinyl alcohol) [16], amphiphilic block copolymer PEA [15], soluble starch [17], heparin [18], chitosan [8,11,12,19], etc. Chitosan, the second most naturally abundant polysaccharide, consists of glucosamine and N-acetyl glucosamine units liked together by -1,4-glucosidic bonds. Due to its biodegradable, biocompatible, non-toxic and antimicrobial characteristics, chitosan is utilized in many areas. For the preparation of silver nanoparticles, chitosan has been used either as a reducing agent [13] or as a stabilizer [11–13]. Although the synthesis of silver nanoparticles in chitosan–acetic acid solution using ␥-ray irradiation has been reported, the irradiation condition was under nitrogen atmosphere [11,12,20]. The present research studies the fabrication of silver nanoparticles in chitosan–aqueous acetic acid solution using ␥-ray irradiation under simple conditions, i.e., air atmosphere. The formation of silver nanoparticles is confirmed by the specific surface plasmon resonance (SPR) band and electron micrograph. The effects of ␥-ray dose (2.5–25.0 kGy), chitosan concentration (0.1 and 0.5%, w/v) and silver nitrate content (0.02–0.10 mmole) on the particle size and particle number are also investigated. In addi-
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tion, the antimicrobial activity of the silver nanoparticles is studied. 2. Experimental methods 2.1. Materials Chitosan (deacetylation degree of 0.95 and molecular weight of ∼700,000 Da) was purchased from the Seafresh Chitosan (Lab) Company Limited, Thailand. Silver nitrate and acetic acid were supplied by Merck, Germany. All chemicals were used as received and without further purification. 2.2. Instruments and equipment Gamma ray irradiation was carried out in a 60 Co Gammacell irradiator with a dose rate of 12 kGy h−1 kindly provided by the Office of Atomic Energy for Peace, Ministry of Science and Technology, Bangkok, Thailand. Ultraviolet and visible (UV–vis) absorption spectra were recorded over a wavelength from 200 to 500 nm by a Helios Gamma Thermo Spectronic spectrometer (England). Transmission electron microscopy (TEM) photographs were taken at an accelerating voltage of 100.0 kV by a Hitachi H-7650 (Hitachi High-Technology Corporation, Japan). Zeta potential was determined at 20 ◦ C by a Malvern Zetasizer (model 3600, Malvern Instruments Ltd., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixed scattering angle of 90◦ . 2.3. Preparation of silver nanoparticles in chitosan solution using -ray irradiation Two concentrations of chitosan solutions (0.1 and 0.5%, w/v) were prepared by stirring chitosan in 1% (v/v) aqueous acetic acid at room temperature overnight. The solutions were then centrifuged to eliminate traces of insoluble fractions. A fresh solution of 50 mM AgNO3 was prepared in 1% (v/v) aqueous acetic acid at room temperature. Chitosan solution (20 mL) was mixed with different amounts of AgNO3 (0.02, 0.04, 0.06, 0.08, and 0.10 mmole). The final volume was adjusted to be 22 mL with 1% (v/v) aqueous acetic acid. The mixture was stirred at room temperature for 10 min and then ␥-ray irradiated in a 60 Co Gammacell irradiator with the dose rate of 12 kGy h−1 . The dose of ␥-ray was varied as 0.0, 2.5, 5.0, 10.0, 15.0, 20.0, and 25.0 kGy. Chitosan solution without the addition of AgNO3 was also prepared in the same manner and used as a control. 2.4. Study on antimicrobial activity of silver nanoparticles Antimicrobial activity of silver nanoparticles was studied by an agar diffusion method using polysaccharide as a film matrix. Silver particles incorporated in polysaccharide-based films were prepared by mixing 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing 0.04 mmole AgNO3 with polysaccharide aqueous solution. The content of silver nanoparticles was varied by changing concentration of the ␥-ray irradiated chitosan solutions containing AgNO3 (0 (control), 10 and 20%,
297
v/v). The mixtures were then cast onto acrylic plates and dried at 45 ◦ C overnight. The dried films were cut into discs of 28 mm diameter followed by sterilization in an autoclave at 121 ◦ C for 15 min. The discs were placed on a surface of nutrient agar (HiMedia Laboratories Pvt. Ltd., India), which had been previously seeded with 20 L of inoculum containing tested bacteria, i.e., Escherichia coli (ATCC 35218, 6.4 × 105 CFU mL−1 ) and Staphylococcus aureus (ATCC 6538, 7.4 × 108 CFU mL−1 ). The plates were incubated at 37 ◦ C for 24 h. The contact areas of the films with agar surface were observed and the diameters of clear zones surrounding the film discs were measured.
3. Results and discussion Conventionally, silver nanoparticles are simply prepared by the reduction of silver salts using chemical reducing agents. Although the chemical reduction is effective, the toxicity of residual reducing agents and the environmental impact should be considered. The present work, thus, focused on the ␥-ray irradiation–reduction under simple conditions, i.e., air atmosphere, to prepare silver nanoparticles. Silver nitrate was applied as a salt precursor, while chitosan was used as a stabilizer. To study the factors affecting formed particle size and number of particles, the ␥-ray dose, initial AgNO3 content, and chitosan concentration were varied. The amount of ␥-ray used was from 2.5 up to 25.0 kGy, initial AgNO3 content was in the range of 0.02–0.10 mmole, and chitosan concentrations were 0.1 and 0.5% (w/v). It should be noted that as chitosan is non-toxic and has been approved by FDA, the silver nanoparticles dispersing in chitosan solution do not need to be separated and/or purified and may be directly used in antibacterial fields. 3.1. Formation of silver particles: physical appearance and mechanism In general, chitosan solution is transparent without color (Fig. 1(a0 )). The solution turns pale yellow after ␥-ray irradiation (Fig. 1(a)). The color intensity increased with increasing ␥-ray dose, especially in the case of 0.5% (w/v) chitosan solution. This might involve the double-bond formation by the chain scission of polysaccharide as reported previously [21,22]. Here, UV–vis spectrophotometry technique was also applied to determine the structural changes of chitosan after ␥-ray irradiation. By comparison with unirradiated chitosan solution (Fig. 2(a)), the ␥-
Fig. 1. Appearances of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v) containing different AgNO3 contents: (a) 0.00 mmole, (b) 0.02 mmole, (c) 0.04 mmole, (d) 0.06 mmole, (e) 0.08 mmole, and (f) 0.10 mmole, after ␥-ray irradiation with various doses from 2.5 to 25.0 kGy. The first column (a0 ) is chitosan solution without the addition of AgNO3 and without ␥-ray irradiation.
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Fig. 2. UV–vis absorption spectra of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), after ␥-ray irradiation with different doses: (a) 0.0 kGy, (b) 2.5 kGy, (c) 5.0 kGy, (d) 10.0 kGy, (e) 15.0 kGy, (f) 20.0 kGy, and (g) 25.0 kGy.
ray irradiated samples gave two new peaks at 260 and 290 nm (Fig. 2(b)–(g)). The absorption band that appeared at 260 nm might corresponds to the C O in COOH group, whereas the one at 290 nm belonged to a terminal carbonyl structure formed at C1 or C4 after the main chain scission of chitosan and hydrogen abstraction followed by the ring opening reaction [23–26]. The absorbance increased with increasing ␥-ray dose up to 20.0 kGy (Fig. 2(b)–(f)) and chitosan concentration. However, at high ␥-ray dose, i.e., 25 kGy, the decomposition and/or conversion of carbonyl and carboxyl species might have taken place resulting in reduced absorbance (Fig. 2(g)). The ␥-ray irradiated chitosan solutions containing AgNO3 exhibit more intense yellow color (Fig. 1(b)–(f)) than those without the addition of AgNO3 (Fig. 1(a)) implying the formation of silver particles [11,20]. This result was also confirmed by UV–vis spectrophotometry (see Section 3.2) and TEM techniques (see Section 3.3). When the initial AgNO3 content and ␥-ray dose increased, the solution color mostly changed from yellow to red-brown. In other words, the color intensity progressed as a function of AgNO3 content and ␥-ray dose. For 0.1% (w/v) chitosan solution after 25.0 kGy ␥-ray irradiation, the maximum color intensity (dark redbrown) appeared when AgNO3 content of 0.04–0.06 mmole was used (Fig. 1(c) and (d), left). This might have resulted from either a large amount of silver particle formation or silver particle aggregation [27]. When the ␥-ray dose was in the range of 10.0–25.0 kGy, the color of 0.1% (w/v) chitosan solutions containing AgNO3 was more intense than that of 0.5% (w/v) chitosan solutions. We speculate that the reduction of silver ions (Ag+ ) to silver particles (Ag0 ) was affected not only by ␥-ray dose, but also by weight ratio of AgNO3 to chitosan. The weight ratio of AgNO3 to chitosan in the case of 0.1% (w/v) chitosan solution was fivefold higher than that of 0.5% (w/v) chitosan solution. Herein, we suspect that ␥-ray irradiation induced both reactions, which are the chain scission of chitosan and the reduction of silver ions to silver particles. For the former, hydroxyl radicals and hydrogen atoms, which are the intermediate products of water radiolysis (Eq. (1)), react with polysaccharide molecules by hydrogen abstraction resulting in the formation of macroradicals localized at various carbon atoms (C1 –C6 ) within the glucosamine unit (Eq. (2)) [23,28]. Only radicals formed at C1 and C4 atoms can undergo rearrangement involving breakage of 1–4 glycosidic bonds (Eq. (3)) and formation of C O species as discussed above. For the latter, the hydrated electron generated by water radiolysis can reduce Ag+ to Ag0 (Eq. (4)) [11,12,29]. The neutral atom, i.e., Ag0 , reacts with Ag+ to form the relatively stabilized Ag clusters as shown in Eqs. (5) and
(6) [30,31]. Such clusters then gather together or absorb the neutral Ag0 to form the silver particles [11,12,30,31]. ioizing
+ • H2 O −→ OH • , e− aq , H , H2 O2 , H2 , H
(1)
OH• (H• ) + R–H → R • (C1 –C6 ) + H2 O(H2 )
(2)
R • (C1 , C4 ) → F1 • + F2 • (chainscission)
(3)
reduction 0 Ag+ + e− aq −→ Ag
(4)
Ag0 + Ag+ → Ag2 +
(5)
Ag2 + + Ag+ → Ag3 2+
(6)
radiation
By considering the structure of chitosan in acidic solution, amino groups were protonated (–NH3 + ). Accordingly, the binding of silver clusters by chitosan may be achieved through the Ag–O bonds as reported in previous studies [12,32,33], the ionic interaction formed between silver ion and carboxyl group on chitosan generated during ␥-ray irradiation (Ag+ . . .COO− ), or the combination thereof. The protonated amino groups (–NH3 + ) at the surface of formed silver particles, thus, promoted the stability of the particles via static repulsion. This speculation is supported by the result from zeta potentiometer, e.g. a positively charged surface with the zeta potential of 40.41 ± 2.50 mV was observed for silver particles formed in 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing 0.04 mmole AgNO3 . It should be pointed out that silver particles formed in 0.5% (w/v) chitosan solution were stable for more than 3 months, whereas those in 0.1% (w/v) chitosan solution were precipitated out within a few weeks. This might be due to a larger number of chitosan chains enveloping the silver particle surface when 0.5% (w/v) chitosan solution was used. 3.2. Formation of silver particles: number of particles UV–vis spectrophotometry technique was applied to confirm the formation of silver particles in the ␥-ray irradiated chitosan solution. Generally, ␥-ray irradiated chitosan solutions exhibit no absorption peak at wavelengths longer than 350 nm (Fig. 2(b)–(g)), while the ones containing AgNO3 show a peak at 401–409 nm corresponding to the surface plasmon resonance band of silver particles (Fig. 3) [34]. An increase in absorbance and a slight shift of SPR band as a function of ␥-ray dose and initial AgNO3 content can be observed in Fig. 3 and are summarized in Figs. 4 and 5.
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Fig. 3. UV–vis absorption spectra of 0.5% (w/v) chitosan solution (in 1% (v/v) aqueous acetic acid solution) containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation with different doses. (A) 20.0 kGy and (B) 25.0 kGy.
Fig. 4. Maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containing various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.
The SPR bands of ␥-ray irradiated chitosan solutions containing AgNO3 appear at the wavelength of 399–417 nm (Fig. 4). A red shift of SPR band (to higher wavelength) was observed when the initial AgNO3 content increased (Fig. 4). This indicated an increase in particle size and/or the formation of silver aggregates [34,35]. It is speculated that the collision frequency of silver particles increased significantly with increasing AgNO3 content resulting in silver particle aggregation. This speculation was also confirmed by TEM
(see Section 3.3). However, the ␥-ray dose hardly affected the SPR band. The amount of silver particles formed related to the absorbance of ␥-ray irradiated chitosan solution containing AgNO3 [11,36]. For low ␥-ray dose, i.e., 2.5 and 5.0 kGy, the absorbance was constant at 2–3 and 6–9 for 2.5 and 5.0 kGy, respectively, though AgNO3 content is varied (Fig. 5). In other words, AgNO3 content hardly affected the absorbance or the number of particles formed at low
Fig. 5. Absorbance at maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v), containing various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.
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␥-ray dose. However, when the ␥-ray doses were 10.0 and 15.0 kGy, the intensity increased up to 0.06 mmole AgNO3 content implying a larger number of silver particles formed for this condition. In addition, the largest number of silver particles formed was obtained when AgNO3 content was 0.08 mmole for ␥-ray dose of 20 kGy and
0.10 mmole for 25 kGy. This might be explained by the formation rate of nuclei being more significant than the growth rate of silver nanocrystal when the AgNO3 contents were less than those specific values, e.g., 0.06 mmole for 10.0 and 15.0 kGy, 0.08 mmole for 20.0 kGy, and 0.10 mmole for 25.0 kGy. The formation of silver
Fig. 6. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v), containing AgNO3 content of 0.10 mmole after ␥-ray irradiation with different doses: (a) 2.5 kGy, (b) 5.0 kGy, (c) 10.0 kGy, (d) 15.0 kGy, (e) 20.0 kGy and (f) 25.0 kGy. Bar is 50 nm.
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Fig. 7. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation with the dose of 25.0 kGy. Bar is 50 nm.
particles increased with increasing ␥-ray dose up to 20 kGy. The reduced number of silver particles formed in 25 kGy ␥-ray irradiated chitosan solution might result from the particles’ aggregation and/or the growth rate of silver nanocrystal over the formation rate of nuclei. In addition, the amount of silver particles formed in 0.1% (w/v) chitosan solutions was not significantly different from that in 0.5% (w/v) chitosan solutions when the same ␥-ray dose was applied.
3.3. Shape and size of silver particles Shape and size of silver particles formed in the ␥-ray irradiated chitosan solutions were observed by TEM. Most particles were spherical with an average diameter of 7–30 nm (Figs. 6 and 7). The size of silver nanoparticles was dependent on ␥-ray dose, initial AgNO3 content, and chitosan concentration.
Fig. 8. Photographs of antimicrobial test results against (A) E. coli and (B) S. aureus of (a) polysaccharide-based film and (b) silver nanoparticles incorporated polysaccharidebased film.
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Table 1 Antimicrobial activity of polysaccharide-based film and silver nanoparticles incorporated polysaccharide-based film against E. coli and S. aureus. Concentration of 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solutions containing 0.04 mmole AgNO3 (%, v/v of polysaccharide aqueous solution)
0 10 20 a
Clear zone (cm)a
E. coli
S. aureus
0.000 ± 0.000 0.308 ± 0.038 0.354 ± 0.019
0.000 ± 0.000 0.079 ± 0.007 0.092 ± 0.036
Reported as mean ± standard deviation (n = 3).
Although, Long et al. revealed that silver nanoparticles could not be formed at the ␥-ray dose of less than 5 kGy [12], the present work found a slightly different result in the formation of metal nanoparticles at low ␥-ray dose, i.e., 2.5 and 5.0 kGy. This formation was affirmable by the appearance of SPR band (see Section 3.2). However, such a formation was incomplete as manifested in Fig. 6(a) and (b). The nanoparticles were maturely formed when the dose was higher than 5.0 kGy. The size of particles increased with increasing ␥-ray dose, e.g., 20, 20, 25, and 30 nm for ␥-ray dose of 10.0, 15.0, 20.0, and 25.0 kGy, respectively, when 0.1% (w/v) chitosan solution was used (Fig. 6A(c)–(f)). For chitosan solution with the concentration of 0.5% (w/v), the result showed same trend. The diameters of particles were 14, 14, 17, and 17, when ␥-ray dose was 10.0, 15.0, 20.0, and 25.0 kGy, respectively (Fig. 6B(c)–(f)). The size of silver nanoparticles was more significantly affected by the initial AgNO3 content than ␥-ray dose. The size increased with increasing initial amount of AgNO3 . For instance, silver particles formed in 25 kGy ␥-ray irradiated 0.1% (w/v) chitosan solution containing different AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and 0.10 mmole had average diameters of 9, 12, 17, 19, and 23 nm, respectively (Fig. 7A). In the same way, the average diameters of silver particles formed in higher chitosan concentration, i.e., 0.5% (w/v) chitosan solution containing AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and 0.10 mmole were 7, 9, 11, 12, and 14 nm, respectively (Fig. 7B). This result corresponded to the one reported by Long et al. [12] and Lei and Fan [15]. The nanoparticles formed in 0.1% (w/v) chitosan solution (Figs. 6A and 7A) were larger than those formed in 0.5% (w/v) chitosan solution (Figs. 6B and 7B). This supported the above results (see Section 3.2). At high concentration of cationic polymer solution, the low collision, low aggregation and/or agglomeration, and strong electrostatic interactions were outstanding; as a result, tiny metal nanoparticles with monodispersity were obtained. This is in accordance with the result of Patakfalvi et al. [16] 3.4. Antimicrobial activity of silver nanoparticles The antimicrobial activity of silver nanoparticles incorporated in polysaccharide-based films against E. coli and S. aureus, which were applied as model Gram-negative and Gram-positive bacteria, respectively, was studied by an agar diffusion method. After 24 h incubation at 37 ◦ C, the contact areas of all films with agar surface turned transparent indicating the inhibition of bacteria growth (Fig. 8). However, clear zones surrounding the film discs were observed only for the films containing silver nanoparticles (Fig. 8(b)). This implies that silver nanoparticles exhibited inhibitory activity against E. coli and S. aureus. The inhibitory efficiency related to the size of the inhibition zone, i.e., the larger the clear area around the film, the higher the inhibitory efficiency. The antimicrobial activity of the film increased with increasing silver nanoparticle content (Table 1). Silver nanoparticles showed higher antimicrobial activity against E. coli than S. aureus (Table 1).
4. Conclusion The preparation of silver nanoparticles was carried out by ␥ray irradiation under simple conditions, i.e., air atmosphere, using chitosan as a stabilizer. The ␥-ray doses of 10–25 kGy were sufficient to achieve maturely formed particles. The obtained particles were spherical with an approximate size of 7–30 nm. The diameter of the particles increased as a function of ␥-ray dose and initial silver nitrate content. Chitosan–aqueous acetic acid solution with the concentration of 0.5% (w/v) was preferred for fabrication of stable, monodispersed, and tiny silver nanoparticles compared to 0.1% (w/v) chitosan solution. The obtained silver nanoparticles inhibited the growth of the model Gram-positive and Gram-negative bacteria. The results suggest that silver nanoparticles dispersed in chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packaging and biomedical applications Acknowledgements The authors thank the Thailand Research Fund (Grant No. MRG5080398) for the financial support. Appreciation is also expressed to the Office of Atomic Energy for Peace, Ministry of Science and Technology, Thailand for its kind allowance to use a 60 Co Gammacell irradiator and Hitachi Hi-Technologies Corporation, Japan for TEM observation. Sincere gratitude goes to Mr. Adrian Hillman for his proof reading. References [1] I. Sondi, B. Salopek-Sondi, J. Colloids Interf. Sci. 275 (2004) 177. [2] Q. Wu, H. Cao, Q. Luan, J. Zhang, Z. Wang, J.H. Warner, A.A.R. Watt, Inorg. Chem. 47 (2008) 5882. [3] L.S. Nair, C.T. Laurencin, J. Biomed. Nanotechnol. 3 (2007) 301. [4] Food casing based on cellulose hydrate with nanoparticles, United States Patent 20080145576. [5] M. Siegrist, M.-E. Cousin, H. Kastenholz, A. Wiek, Appetite 49 (2007) 459. [6] K. Dhermendra, J.B. Tiwari, P. Sen, World Appl. Sci. J. 3 (2008) 417. [7] H. Jiang, S. Manolache, A.C.L. Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004) 1411. [8] H. Huang, Q. Yuan, X. Yang, Colloids Surf. B: Biointerf. 39 (2004) 31. [9] C.R.K. Rao, D.C. Trivedi, Synth. Met. 155 (2005) 324. [10] W. Zhang, X. Qiao, J. Chen, H. Wang, J. Colloids Interf. Sci. 302 (2006) 370. [11] P. Chen, L. Song, Y. Liu, Y. Fang, Radiat. Phys. Chem. 76 (2007) 1165. [12] D. Long, G. Wu, S. Chen, Radiat. Phys. Chem. 76 (2007) 1126. [13] D.M. Cheng, X.D. Zhou, H.B. Xia, H.S.O. Chan, Chem. Mater. 17 (2005) 3578. [14] M. Tsuji, Y. Nishizawa, K. Matsumoto, N. Miyamae, T. Tsuji, X. Zhang, Colloids Surf. A: Physicochem. Eng. Aspects 293 (2007) 185. [15] Z. Lei, Y. Fan, Mater. Lett. 60 (2006) 2256. [16] R. Patakfalvi, Z. Viranyi, I. Dekany, Colloids Polym. Sci. 283 (2004) 299. [17] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V. Varadarajan, Carbohydr. Res. 341 (2006) 2012. [18] H. Huang, X. Yang, Carbohydr. Res. 339 (2004) 2627. [19] D. Wei, W. Qian, Colloids Surf. B: Biointerf. 62 (2008) 136. [20] X. Xu, Y. Yin, X. Ge, H. Wu, Z. Zhang, Mater. Lett. 37 (1998) 354. [21] N. Nagasawa, H. Mitomo, F. Yoshii, T. Kume, Polym. Degrad. Stabil. 69 (2000) 279. [22] W.S. Choi, K.J. Ahn, D.W. Lee, M.W. Byun, H.J. Park, Polym. Degrad. Stabil. 78 (2002) 533. [23] P. Ulanski, J.M. Rosiak, Radiat. Phys. Chem. 39 (1992) 53. [24] B. Kang, Y.-D. Dai, H.-Q. Zhang, D. Chen, Polym. Degrad. Stabil. 92 (2007) 359. [25] R. Yoksan, M. Akashi, S. Biramontri, S. Chirachanchai, Biomacromolecules 2 (2001) 1038. [26] R. Czechowska-Biskup, B. Rokita, S. Lotfy, P. Ulanski, J.M. Rosiak, Carbohydr. Polym. 60 (2005) 175. [27] J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S. Schultz, J. Chem. Phys. 116 (2002) 6755. [28] R. Yoksan, M. Akashi, M. Miyata, S. Chirachanchai, Radiat. Res. 161 (2004) 471. [29] Y. Zhu, Y. Qian, X. Li, M. Zhang, Chem. Commun. 12 (1997) 1081. [30] E. Janata, A. Henglein, B.G. Ershov, J. Phys. Chem. 98 (1994) 10888. [31] E. Janata, J. Phys. Chem. B 107 (2003) 7334. [32] S.P. Chen, G.Z. Wu, H.Y. Zeng, Carbohydr. Polym. 60 (2005) 33. [33] H.H. Huang, X.P. Ni, G.L. Loy, C.H. Chew, K.L. Tan, F.C. Loh, J.F. Deng, G.Q. Xu, Langmuir 12 (1996) 909. [34] A. Kumar, H. Joshi, R. Pasricha, A.B. Mandale, M. Sastry, J. Colloids Interf. Sci. 264 (2003) 396. [35] J.P. Zhang, P. Chen, C.H. Sun, X.J. Hu, Appl. Catal. A: Gen. 266 (2004) 49. [36] Z.Q. Zhang, R.C. Patel, R. Kothari, C.P. Johnson, S.E. Friberg, P.A. Aikens, J. Phys. Chem. B 104 (2000) 1176.
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Silver nanoparticles dispersing in chitosan solution: Preparation by ␥-ray irradiation and their antimicrobial activities Rangrong Yoksan a,b,∗ , Suwabun Chirachanchai c a
Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, Thailand Division of Physico-Chemical Processing Technology, Faculty of Agro-Industry, Kasesart University, Bangkok 10900, Thailand c The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand b
a r t i c l e
i n f o
Article history: Received 25 September 2008 Received in revised form 16 November 2008 Accepted 1 December 2008 Keywords: Silver Nanoparticle Chitosan Irradiation Transmission electron microscopy (TEM)
a b s t r a c t Silver nanoparticles were prepared by ␥-ray irradiation–reduction under simple conditions, i.e., air atmosphere, using chitosan as a stabilizer. The nanoparticles were spherical with an average size of 7–30 nm as observed from TEM. The size decreased when chitosan concentration increased, while it increased with increasing ␥-ray dose and initial silver nitrate content. The obtained silver nanoparticles dispersed in a 0.5% (w/v) ␥-ray irradiated chitosan–aqueous acetic acid solution were stable for more than 3 months without tendency to precipitate. The silver nanoparticles exhibited antimicrobial activities against Escherichia coli and Staphylococcus aureus. The results suggest that silver nanoparticles dispersed in chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packaging and biomedical applications. © 2008 Elsevier B.V. All rights reserved.
1. Introduction For many decades, silver nanoparticles have been recognized as an antimicrobial agent [1] and used in many fields including biomedical [2,3], food packaging [4,5], waste water treatment [6], etc. The antimicrobial activities of silver nanoparticles were found to relate to their shapes and sizes [7]. Many attempts have been made to synthesize the silver nanoparticles with controllable shape, size, and size distribution. In most cases, the system for fabricating silver nanoparticles consists of salt precursor, reducing agent, and stabilizer. Silver nitrate (AgNO3 ) is usually applied as the salt precursor. The reduction of silver ions (Ag+ ) to silver particles (Ag0 ) is conventionally achieved by a chemical method using reducing agents such as NaBH4 [8], formamide [9], dimethylformamide [9], triethanolamine [9], hydrazine [10], etc. Although this method is simple and effective, the biological toxicity and the environmental hazard of the residual reducing agent are problems. Recently, reductions by other techniques such as ␥-ray irradiation [11,12], UV irradiation [13], microwave [14], and ultrasonic [15] have been alternative ways to overcome those problems and to
∗ Corresponding author at: Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, Thailand. Tel.: +66 2 562 5097; fax: +66 2 562 5092. E-mail addresses: [email protected], [email protected] (R. Yoksan). 0254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2008.12.001
eliminate the purification step or the removal of reducing agent step. To prevent the aggregation of formed metal nanoparticles, various types of stabilizers are used such as long-chain fatty acids (stearic, palmitic, and lauric acids) [9], lauryl amine, poly(vinyl pyrrolidone) [16], poly(vinyl alcohol) [16], amphiphilic block copolymer PEA [15], soluble starch [17], heparin [18], chitosan [8,11,12,19], etc. Chitosan, the second most naturally abundant polysaccharide, consists of glucosamine and N-acetyl glucosamine units liked together by -1,4-glucosidic bonds. Due to its biodegradable, biocompatible, non-toxic and antimicrobial characteristics, chitosan is utilized in many areas. For the preparation of silver nanoparticles, chitosan has been used either as a reducing agent [13] or as a stabilizer [11–13]. Although the synthesis of silver nanoparticles in chitosan–acetic acid solution using ␥-ray irradiation has been reported, the irradiation condition was under nitrogen atmosphere [11,12,20]. The present research studies the fabrication of silver nanoparticles in chitosan–aqueous acetic acid solution using ␥-ray irradiation under simple conditions, i.e., air atmosphere. The formation of silver nanoparticles is confirmed by the specific surface plasmon resonance (SPR) band and electron micrograph. The effects of ␥-ray dose (2.5–25.0 kGy), chitosan concentration (0.1 and 0.5%, w/v) and silver nitrate content (0.02–0.10 mmole) on the particle size and particle number are also investigated. In addi-
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tion, the antimicrobial activity of the silver nanoparticles is studied. 2. Experimental methods 2.1. Materials Chitosan (deacetylation degree of 0.95 and molecular weight of ∼700,000 Da) was purchased from the Seafresh Chitosan (Lab) Company Limited, Thailand. Silver nitrate and acetic acid were supplied by Merck, Germany. All chemicals were used as received and without further purification. 2.2. Instruments and equipment Gamma ray irradiation was carried out in a 60 Co Gammacell irradiator with a dose rate of 12 kGy h−1 kindly provided by the Office of Atomic Energy for Peace, Ministry of Science and Technology, Bangkok, Thailand. Ultraviolet and visible (UV–vis) absorption spectra were recorded over a wavelength from 200 to 500 nm by a Helios Gamma Thermo Spectronic spectrometer (England). Transmission electron microscopy (TEM) photographs were taken at an accelerating voltage of 100.0 kV by a Hitachi H-7650 (Hitachi High-Technology Corporation, Japan). Zeta potential was determined at 20 ◦ C by a Malvern Zetasizer (model 3600, Malvern Instruments Ltd., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixed scattering angle of 90◦ . 2.3. Preparation of silver nanoparticles in chitosan solution using -ray irradiation Two concentrations of chitosan solutions (0.1 and 0.5%, w/v) were prepared by stirring chitosan in 1% (v/v) aqueous acetic acid at room temperature overnight. The solutions were then centrifuged to eliminate traces of insoluble fractions. A fresh solution of 50 mM AgNO3 was prepared in 1% (v/v) aqueous acetic acid at room temperature. Chitosan solution (20 mL) was mixed with different amounts of AgNO3 (0.02, 0.04, 0.06, 0.08, and 0.10 mmole). The final volume was adjusted to be 22 mL with 1% (v/v) aqueous acetic acid. The mixture was stirred at room temperature for 10 min and then ␥-ray irradiated in a 60 Co Gammacell irradiator with the dose rate of 12 kGy h−1 . The dose of ␥-ray was varied as 0.0, 2.5, 5.0, 10.0, 15.0, 20.0, and 25.0 kGy. Chitosan solution without the addition of AgNO3 was also prepared in the same manner and used as a control. 2.4. Study on antimicrobial activity of silver nanoparticles Antimicrobial activity of silver nanoparticles was studied by an agar diffusion method using polysaccharide as a film matrix. Silver particles incorporated in polysaccharide-based films were prepared by mixing 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing 0.04 mmole AgNO3 with polysaccharide aqueous solution. The content of silver nanoparticles was varied by changing concentration of the ␥-ray irradiated chitosan solutions containing AgNO3 (0 (control), 10 and 20%,
297
v/v). The mixtures were then cast onto acrylic plates and dried at 45 ◦ C overnight. The dried films were cut into discs of 28 mm diameter followed by sterilization in an autoclave at 121 ◦ C for 15 min. The discs were placed on a surface of nutrient agar (HiMedia Laboratories Pvt. Ltd., India), which had been previously seeded with 20 L of inoculum containing tested bacteria, i.e., Escherichia coli (ATCC 35218, 6.4 × 105 CFU mL−1 ) and Staphylococcus aureus (ATCC 6538, 7.4 × 108 CFU mL−1 ). The plates were incubated at 37 ◦ C for 24 h. The contact areas of the films with agar surface were observed and the diameters of clear zones surrounding the film discs were measured.
3. Results and discussion Conventionally, silver nanoparticles are simply prepared by the reduction of silver salts using chemical reducing agents. Although the chemical reduction is effective, the toxicity of residual reducing agents and the environmental impact should be considered. The present work, thus, focused on the ␥-ray irradiation–reduction under simple conditions, i.e., air atmosphere, to prepare silver nanoparticles. Silver nitrate was applied as a salt precursor, while chitosan was used as a stabilizer. To study the factors affecting formed particle size and number of particles, the ␥-ray dose, initial AgNO3 content, and chitosan concentration were varied. The amount of ␥-ray used was from 2.5 up to 25.0 kGy, initial AgNO3 content was in the range of 0.02–0.10 mmole, and chitosan concentrations were 0.1 and 0.5% (w/v). It should be noted that as chitosan is non-toxic and has been approved by FDA, the silver nanoparticles dispersing in chitosan solution do not need to be separated and/or purified and may be directly used in antibacterial fields. 3.1. Formation of silver particles: physical appearance and mechanism In general, chitosan solution is transparent without color (Fig. 1(a0 )). The solution turns pale yellow after ␥-ray irradiation (Fig. 1(a)). The color intensity increased with increasing ␥-ray dose, especially in the case of 0.5% (w/v) chitosan solution. This might involve the double-bond formation by the chain scission of polysaccharide as reported previously [21,22]. Here, UV–vis spectrophotometry technique was also applied to determine the structural changes of chitosan after ␥-ray irradiation. By comparison with unirradiated chitosan solution (Fig. 2(a)), the ␥-
Fig. 1. Appearances of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v) containing different AgNO3 contents: (a) 0.00 mmole, (b) 0.02 mmole, (c) 0.04 mmole, (d) 0.06 mmole, (e) 0.08 mmole, and (f) 0.10 mmole, after ␥-ray irradiation with various doses from 2.5 to 25.0 kGy. The first column (a0 ) is chitosan solution without the addition of AgNO3 and without ␥-ray irradiation.
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Fig. 2. UV–vis absorption spectra of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), after ␥-ray irradiation with different doses: (a) 0.0 kGy, (b) 2.5 kGy, (c) 5.0 kGy, (d) 10.0 kGy, (e) 15.0 kGy, (f) 20.0 kGy, and (g) 25.0 kGy.
ray irradiated samples gave two new peaks at 260 and 290 nm (Fig. 2(b)–(g)). The absorption band that appeared at 260 nm might corresponds to the C O in COOH group, whereas the one at 290 nm belonged to a terminal carbonyl structure formed at C1 or C4 after the main chain scission of chitosan and hydrogen abstraction followed by the ring opening reaction [23–26]. The absorbance increased with increasing ␥-ray dose up to 20.0 kGy (Fig. 2(b)–(f)) and chitosan concentration. However, at high ␥-ray dose, i.e., 25 kGy, the decomposition and/or conversion of carbonyl and carboxyl species might have taken place resulting in reduced absorbance (Fig. 2(g)). The ␥-ray irradiated chitosan solutions containing AgNO3 exhibit more intense yellow color (Fig. 1(b)–(f)) than those without the addition of AgNO3 (Fig. 1(a)) implying the formation of silver particles [11,20]. This result was also confirmed by UV–vis spectrophotometry (see Section 3.2) and TEM techniques (see Section 3.3). When the initial AgNO3 content and ␥-ray dose increased, the solution color mostly changed from yellow to red-brown. In other words, the color intensity progressed as a function of AgNO3 content and ␥-ray dose. For 0.1% (w/v) chitosan solution after 25.0 kGy ␥-ray irradiation, the maximum color intensity (dark redbrown) appeared when AgNO3 content of 0.04–0.06 mmole was used (Fig. 1(c) and (d), left). This might have resulted from either a large amount of silver particle formation or silver particle aggregation [27]. When the ␥-ray dose was in the range of 10.0–25.0 kGy, the color of 0.1% (w/v) chitosan solutions containing AgNO3 was more intense than that of 0.5% (w/v) chitosan solutions. We speculate that the reduction of silver ions (Ag+ ) to silver particles (Ag0 ) was affected not only by ␥-ray dose, but also by weight ratio of AgNO3 to chitosan. The weight ratio of AgNO3 to chitosan in the case of 0.1% (w/v) chitosan solution was fivefold higher than that of 0.5% (w/v) chitosan solution. Herein, we suspect that ␥-ray irradiation induced both reactions, which are the chain scission of chitosan and the reduction of silver ions to silver particles. For the former, hydroxyl radicals and hydrogen atoms, which are the intermediate products of water radiolysis (Eq. (1)), react with polysaccharide molecules by hydrogen abstraction resulting in the formation of macroradicals localized at various carbon atoms (C1 –C6 ) within the glucosamine unit (Eq. (2)) [23,28]. Only radicals formed at C1 and C4 atoms can undergo rearrangement involving breakage of 1–4 glycosidic bonds (Eq. (3)) and formation of C O species as discussed above. For the latter, the hydrated electron generated by water radiolysis can reduce Ag+ to Ag0 (Eq. (4)) [11,12,29]. The neutral atom, i.e., Ag0 , reacts with Ag+ to form the relatively stabilized Ag clusters as shown in Eqs. (5) and
(6) [30,31]. Such clusters then gather together or absorb the neutral Ag0 to form the silver particles [11,12,30,31]. ioizing
+ • H2 O −→ OH • , e− aq , H , H2 O2 , H2 , H
(1)
OH• (H• ) + R–H → R • (C1 –C6 ) + H2 O(H2 )
(2)
R • (C1 , C4 ) → F1 • + F2 • (chainscission)
(3)
reduction 0 Ag+ + e− aq −→ Ag
(4)
Ag0 + Ag+ → Ag2 +
(5)
Ag2 + + Ag+ → Ag3 2+
(6)
radiation
By considering the structure of chitosan in acidic solution, amino groups were protonated (–NH3 + ). Accordingly, the binding of silver clusters by chitosan may be achieved through the Ag–O bonds as reported in previous studies [12,32,33], the ionic interaction formed between silver ion and carboxyl group on chitosan generated during ␥-ray irradiation (Ag+ . . .COO− ), or the combination thereof. The protonated amino groups (–NH3 + ) at the surface of formed silver particles, thus, promoted the stability of the particles via static repulsion. This speculation is supported by the result from zeta potentiometer, e.g. a positively charged surface with the zeta potential of 40.41 ± 2.50 mV was observed for silver particles formed in 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing 0.04 mmole AgNO3 . It should be pointed out that silver particles formed in 0.5% (w/v) chitosan solution were stable for more than 3 months, whereas those in 0.1% (w/v) chitosan solution were precipitated out within a few weeks. This might be due to a larger number of chitosan chains enveloping the silver particle surface when 0.5% (w/v) chitosan solution was used. 3.2. Formation of silver particles: number of particles UV–vis spectrophotometry technique was applied to confirm the formation of silver particles in the ␥-ray irradiated chitosan solution. Generally, ␥-ray irradiated chitosan solutions exhibit no absorption peak at wavelengths longer than 350 nm (Fig. 2(b)–(g)), while the ones containing AgNO3 show a peak at 401–409 nm corresponding to the surface plasmon resonance band of silver particles (Fig. 3) [34]. An increase in absorbance and a slight shift of SPR band as a function of ␥-ray dose and initial AgNO3 content can be observed in Fig. 3 and are summarized in Figs. 4 and 5.
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Fig. 3. UV–vis absorption spectra of 0.5% (w/v) chitosan solution (in 1% (v/v) aqueous acetic acid solution) containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation with different doses. (A) 20.0 kGy and (B) 25.0 kGy.
Fig. 4. Maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containing various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.
The SPR bands of ␥-ray irradiated chitosan solutions containing AgNO3 appear at the wavelength of 399–417 nm (Fig. 4). A red shift of SPR band (to higher wavelength) was observed when the initial AgNO3 content increased (Fig. 4). This indicated an increase in particle size and/or the formation of silver aggregates [34,35]. It is speculated that the collision frequency of silver particles increased significantly with increasing AgNO3 content resulting in silver particle aggregation. This speculation was also confirmed by TEM
(see Section 3.3). However, the ␥-ray dose hardly affected the SPR band. The amount of silver particles formed related to the absorbance of ␥-ray irradiated chitosan solution containing AgNO3 [11,36]. For low ␥-ray dose, i.e., 2.5 and 5.0 kGy, the absorbance was constant at 2–3 and 6–9 for 2.5 and 5.0 kGy, respectively, though AgNO3 content is varied (Fig. 5). In other words, AgNO3 content hardly affected the absorbance or the number of particles formed at low
Fig. 5. Absorbance at maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v), containing various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.
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␥-ray dose. However, when the ␥-ray doses were 10.0 and 15.0 kGy, the intensity increased up to 0.06 mmole AgNO3 content implying a larger number of silver particles formed for this condition. In addition, the largest number of silver particles formed was obtained when AgNO3 content was 0.08 mmole for ␥-ray dose of 20 kGy and
0.10 mmole for 25 kGy. This might be explained by the formation rate of nuclei being more significant than the growth rate of silver nanocrystal when the AgNO3 contents were less than those specific values, e.g., 0.06 mmole for 10.0 and 15.0 kGy, 0.08 mmole for 20.0 kGy, and 0.10 mmole for 25.0 kGy. The formation of silver
Fig. 6. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v), containing AgNO3 content of 0.10 mmole after ␥-ray irradiation with different doses: (a) 2.5 kGy, (b) 5.0 kGy, (c) 10.0 kGy, (d) 15.0 kGy, (e) 20.0 kGy and (f) 25.0 kGy. Bar is 50 nm.
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Fig. 7. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation with the dose of 25.0 kGy. Bar is 50 nm.
particles increased with increasing ␥-ray dose up to 20 kGy. The reduced number of silver particles formed in 25 kGy ␥-ray irradiated chitosan solution might result from the particles’ aggregation and/or the growth rate of silver nanocrystal over the formation rate of nuclei. In addition, the amount of silver particles formed in 0.1% (w/v) chitosan solutions was not significantly different from that in 0.5% (w/v) chitosan solutions when the same ␥-ray dose was applied.
3.3. Shape and size of silver particles Shape and size of silver particles formed in the ␥-ray irradiated chitosan solutions were observed by TEM. Most particles were spherical with an average diameter of 7–30 nm (Figs. 6 and 7). The size of silver nanoparticles was dependent on ␥-ray dose, initial AgNO3 content, and chitosan concentration.
Fig. 8. Photographs of antimicrobial test results against (A) E. coli and (B) S. aureus of (a) polysaccharide-based film and (b) silver nanoparticles incorporated polysaccharidebased film.
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Table 1 Antimicrobial activity of polysaccharide-based film and silver nanoparticles incorporated polysaccharide-based film against E. coli and S. aureus. Concentration of 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solutions containing 0.04 mmole AgNO3 (%, v/v of polysaccharide aqueous solution)
0 10 20 a
Clear zone (cm)a
E. coli
S. aureus
0.000 ± 0.000 0.308 ± 0.038 0.354 ± 0.019
0.000 ± 0.000 0.079 ± 0.007 0.092 ± 0.036
Reported as mean ± standard deviation (n = 3).
Although, Long et al. revealed that silver nanoparticles could not be formed at the ␥-ray dose of less than 5 kGy [12], the present work found a slightly different result in the formation of metal nanoparticles at low ␥-ray dose, i.e., 2.5 and 5.0 kGy. This formation was affirmable by the appearance of SPR band (see Section 3.2). However, such a formation was incomplete as manifested in Fig. 6(a) and (b). The nanoparticles were maturely formed when the dose was higher than 5.0 kGy. The size of particles increased with increasing ␥-ray dose, e.g., 20, 20, 25, and 30 nm for ␥-ray dose of 10.0, 15.0, 20.0, and 25.0 kGy, respectively, when 0.1% (w/v) chitosan solution was used (Fig. 6A(c)–(f)). For chitosan solution with the concentration of 0.5% (w/v), the result showed same trend. The diameters of particles were 14, 14, 17, and 17, when ␥-ray dose was 10.0, 15.0, 20.0, and 25.0 kGy, respectively (Fig. 6B(c)–(f)). The size of silver nanoparticles was more significantly affected by the initial AgNO3 content than ␥-ray dose. The size increased with increasing initial amount of AgNO3 . For instance, silver particles formed in 25 kGy ␥-ray irradiated 0.1% (w/v) chitosan solution containing different AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and 0.10 mmole had average diameters of 9, 12, 17, 19, and 23 nm, respectively (Fig. 7A). In the same way, the average diameters of silver particles formed in higher chitosan concentration, i.e., 0.5% (w/v) chitosan solution containing AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and 0.10 mmole were 7, 9, 11, 12, and 14 nm, respectively (Fig. 7B). This result corresponded to the one reported by Long et al. [12] and Lei and Fan [15]. The nanoparticles formed in 0.1% (w/v) chitosan solution (Figs. 6A and 7A) were larger than those formed in 0.5% (w/v) chitosan solution (Figs. 6B and 7B). This supported the above results (see Section 3.2). At high concentration of cationic polymer solution, the low collision, low aggregation and/or agglomeration, and strong electrostatic interactions were outstanding; as a result, tiny metal nanoparticles with monodispersity were obtained. This is in accordance with the result of Patakfalvi et al. [16] 3.4. Antimicrobial activity of silver nanoparticles The antimicrobial activity of silver nanoparticles incorporated in polysaccharide-based films against E. coli and S. aureus, which were applied as model Gram-negative and Gram-positive bacteria, respectively, was studied by an agar diffusion method. After 24 h incubation at 37 ◦ C, the contact areas of all films with agar surface turned transparent indicating the inhibition of bacteria growth (Fig. 8). However, clear zones surrounding the film discs were observed only for the films containing silver nanoparticles (Fig. 8(b)). This implies that silver nanoparticles exhibited inhibitory activity against E. coli and S. aureus. The inhibitory efficiency related to the size of the inhibition zone, i.e., the larger the clear area around the film, the higher the inhibitory efficiency. The antimicrobial activity of the film increased with increasing silver nanoparticle content (Table 1). Silver nanoparticles showed higher antimicrobial activity against E. coli than S. aureus (Table 1).
4. Conclusion The preparation of silver nanoparticles was carried out by ␥ray irradiation under simple conditions, i.e., air atmosphere, using chitosan as a stabilizer. The ␥-ray doses of 10–25 kGy were sufficient to achieve maturely formed particles. The obtained particles were spherical with an approximate size of 7–30 nm. The diameter of the particles increased as a function of ␥-ray dose and initial silver nitrate content. Chitosan–aqueous acetic acid solution with the concentration of 0.5% (w/v) was preferred for fabrication of stable, monodispersed, and tiny silver nanoparticles compared to 0.1% (w/v) chitosan solution. The obtained silver nanoparticles inhibited the growth of the model Gram-positive and Gram-negative bacteria. The results suggest that silver nanoparticles dispersed in chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packaging and biomedical applications Acknowledgements The authors thank the Thailand Research Fund (Grant No. MRG5080398) for the financial support. Appreciation is also expressed to the Office of Atomic Energy for Peace, Ministry of Science and Technology, Thailand for its kind allowance to use a 60 Co Gammacell irradiator and Hitachi Hi-Technologies Corporation, Japan for TEM observation. Sincere gratitude goes to Mr. Adrian Hillman for his proof reading. References [1] I. Sondi, B. Salopek-Sondi, J. Colloids Interf. Sci. 275 (2004) 177. [2] Q. Wu, H. Cao, Q. Luan, J. Zhang, Z. Wang, J.H. Warner, A.A.R. Watt, Inorg. Chem. 47 (2008) 5882. [3] L.S. Nair, C.T. Laurencin, J. Biomed. Nanotechnol. 3 (2007) 301. [4] Food casing based on cellulose hydrate with nanoparticles, United States Patent 20080145576. [5] M. Siegrist, M.-E. Cousin, H. Kastenholz, A. Wiek, Appetite 49 (2007) 459. [6] K. Dhermendra, J.B. Tiwari, P. Sen, World Appl. Sci. J. 3 (2008) 417. [7] H. Jiang, S. Manolache, A.C.L. Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004) 1411. [8] H. Huang, Q. Yuan, X. Yang, Colloids Surf. B: Biointerf. 39 (2004) 31. [9] C.R.K. Rao, D.C. Trivedi, Synth. Met. 155 (2005) 324. [10] W. Zhang, X. Qiao, J. Chen, H. Wang, J. Colloids Interf. Sci. 302 (2006) 370. [11] P. Chen, L. Song, Y. Liu, Y. Fang, Radiat. Phys. Chem. 76 (2007) 1165. [12] D. Long, G. Wu, S. Chen, Radiat. Phys. Chem. 76 (2007) 1126. [13] D.M. Cheng, X.D. Zhou, H.B. Xia, H.S.O. Chan, Chem. Mater. 17 (2005) 3578. [14] M. Tsuji, Y. Nishizawa, K. Matsumoto, N. Miyamae, T. Tsuji, X. Zhang, Colloids Surf. A: Physicochem. Eng. Aspects 293 (2007) 185. [15] Z. Lei, Y. Fan, Mater. Lett. 60 (2006) 2256. [16] R. Patakfalvi, Z. Viranyi, I. Dekany, Colloids Polym. Sci. 283 (2004) 299. [17] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V. Varadarajan, Carbohydr. Res. 341 (2006) 2012. [18] H. Huang, X. Yang, Carbohydr. Res. 339 (2004) 2627. [19] D. Wei, W. Qian, Colloids Surf. B: Biointerf. 62 (2008) 136. [20] X. Xu, Y. Yin, X. Ge, H. Wu, Z. Zhang, Mater. Lett. 37 (1998) 354. [21] N. Nagasawa, H. Mitomo, F. Yoshii, T. Kume, Polym. Degrad. Stabil. 69 (2000) 279. [22] W.S. Choi, K.J. Ahn, D.W. Lee, M.W. Byun, H.J. Park, Polym. Degrad. Stabil. 78 (2002) 533. [23] P. Ulanski, J.M. Rosiak, Radiat. Phys. Chem. 39 (1992) 53. [24] B. Kang, Y.-D. Dai, H.-Q. Zhang, D. Chen, Polym. Degrad. Stabil. 92 (2007) 359. [25] R. Yoksan, M. Akashi, S. Biramontri, S. Chirachanchai, Biomacromolecules 2 (2001) 1038. [26] R. Czechowska-Biskup, B. Rokita, S. Lotfy, P. Ulanski, J.M. Rosiak, Carbohydr. Polym. 60 (2005) 175. [27] J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S. Schultz, J. Chem. Phys. 116 (2002) 6755. [28] R. Yoksan, M. Akashi, M. Miyata, S. Chirachanchai, Radiat. Res. 161 (2004) 471. [29] Y. Zhu, Y. Qian, X. Li, M. Zhang, Chem. Commun. 12 (1997) 1081. [30] E. Janata, A. Henglein, B.G. Ershov, J. Phys. Chem. 98 (1994) 10888. [31] E. Janata, J. Phys. Chem. B 107 (2003) 7334. [32] S.P. Chen, G.Z. Wu, H.Y. Zeng, Carbohydr. Polym. 60 (2005) 33. [33] H.H. Huang, X.P. Ni, G.L. Loy, C.H. Chew, K.L. Tan, F.C. Loh, J.F. Deng, G.Q. Xu, Langmuir 12 (1996) 909. [34] A. Kumar, H. Joshi, R. Pasricha, A.B. Mandale, M. Sastry, J. Colloids Interf. Sci. 264 (2003) 396. [35] J.P. Zhang, P. Chen, C.H. Sun, X.J. Hu, Appl. Catal. A: Gen. 266 (2004) 49. [36] Z.Q. Zhang, R.C. Patel, R. Kothari, C.P. Johnson, S.E. Friberg, P.A. Aikens, J. Phys. Chem. B 104 (2000) 1176.