J Nanopart Res (2011) 13:2525–2532 DOI 10.1007/s11051-010-0145-6
RESEARCH PAPER
Synthesis and characterization of silver nanoparticles: effect on phytopathogen Colletotrichum gloesporioides Miguel A. Aguilar-Me´ndez • Eduardo San Martı´n-Martı´nez Lesli Ortega-Arroyo • Georgina Cobia´n-Portillo • Esther Sa´nchez-Espı´ndola
•
Received: 26 July 2010 / Accepted: 8 November 2010 / Published online: 20 November 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Colloidal silver nanoparticles were synthesized by reducing silver nitrate solutions with glucose, in the presence of gelatin as capping agent. The obtained nanoparticles were characterized by means of UV–Vis spectroscopy, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The response surface methodology (RSM) was also used to determine the influence of the variables on the size of the nanoparticles. The antifungal activity of the silver nanoparticles was evaluated on the phytopathogen Colletotrichum gloesporioides, which causes anthracnose in a wide range of fruits. The UV– Vis spectra indicated the formation of silver nanoparticles preferably spherical and of relatively small size (\20 nm). The above-mentioned was confirmed by M. A. Aguilar-Me´ndez (&) E. San Martı´n-Martı´nez L. Ortega-Arroyo Instituto Polite´cnico Nacional, Centro de Investigacio´n en Ciencia Aplicada y Tecnologı´a Avanzada, Legaria 694, Colonia Irrigacio´n 11500 Me´xico, DF, Me´xico e-mail:
[email protected] G. Cobia´n-Portillo Instituto Polite´cnico Nacional, Centro Interdisciplinario de Investigacio´n para el Desarrollo Integral Regional, Hornos 1003, Colonia Nochebuena, 71230 Oaxaca, Me´xico E. Sa´nchez-Espı´ndola Instituto Polite´cnico Nacional, Escuela Nacional de Ciencias Biolo´gicas, Prolongacio´n Manuel M. Carpio s/n, esq. Plan de Ayala, Colonia Santo Toma´s, 11340 Me´xico, DF, Me´xico
TEM, observing a size distribution of 5–24 nm. According to RSM the synthesis variables influenced on the size of the silver nanoparticles. By means of FTIR spectroscopy it was determined that gelatin, through their amide and hydroxyl groups, interacts with nanoparticles preventing their agglomeration. The growth of C. gloesporioides in the presence of silver nanoparticles was significantly delayed in a dose dependent manner. Keywords Silver nanoparticles Colletotrichum gloesporiodes Gelatin Antifungal activity Antimicrobial
Introduction Several metals at nanometer scale present antimicrobial properties mainly due to their large surface area (Azeredo 2009). This fact has promoted the study of metal nanoparticles with the purpose of using them as new antimicrobial agents (Sondi and Salopek-Sondi 2004). Among the inorganic antimicrobial agents, silver has been widely used since ancient times to fight infections and control microbial contamination (Pal et al. 2007). The bactericidal effect of silver ions is well known, but the mechanism is not totally clear. Some theories have been developed to explain the inhibitory effect of silver ions and metallic silver on microorganisms (Cho et al. 2005). It is believed that silver ions interact strongly with the thiol groups of vital enzymes, causing their inactivation (Feng et al. 2000). It is also possible that DNA from
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bacteria treated with nanoparticles, loses its ability to replicate due to the affinity of silver to bind with phosphorylated and sulfur groups (Pal et al. 2007). Other studies have reported that silver ions cause irreversible structural changes in the bacterial cell membrane, affecting drastically the permeability and respiration functions (Cho et al. 2005; Morones et al. 2005). The effect of silver nanoparticles against several types of bacteria has been extensively studied; however, there is limited information about the antifungal activity on phytopathogenic fungi (Panacek et al. 2009). Petica et al. (2008) reported only a fungistatic effect of silver nanoparticles against Aspergillius, Penicillium, and Trichoderma species. On the other hand, Kim et al. (2009) observed a significant inhibition in the growth of the fungus Raffaelea sp. when treated with silver nanoparticles. They also reported that the inhibition was more efficient as the concentration of nanoparticles increased. It is estimated that about 20–25% of the harvested fruits and vegetables are decayed by pathogens during postharvest handling (Sharma et al. 2009). One of these pathogens is C. gloesporioides which causes anthracnose in a wide range of fruits, such as apple, avocado, mango, papaya, etc. Anthracnose lesions, for example in strawberry, develop as tan or light brown, circular, sunken lesions on ripe or ripening fruit. Generally, the symptoms become evident only during the postharvest period. Anthracnose is controlled principally by application of synthetic fungicides during the postharvest period (Mun˜oz et al. 2009). However, the indiscriminate use of them has caused the emergence of resistant strains. Also the residue levels of the chemicals may represent a serious problem to the human health (Gamagae et al. 2003). The use of metal nanoparticles as new antimicrobial agents could represent a viable alternative to delay or inhibit the growth of many pathogens species. The aim of this work was to synthesize silver nanoparticles by a chemical reduction method and evaluate their effect on the growth of C. gloesporioides.
Experimental Reagents Silver nitrate (AgNO3) was acquired from SigmaAldrich, USA. Glucose and sodium hydroxide (NaOH)
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were obtained from Baker, Mexico. Gelatin was supplied by Gardhal, Mexico. Deionized water (18 MX cm-1) was used throughout the experiments. Synthesis of silver nanoparticles In a typical experiment, an aqueous solution of gelatin–glucose was prepared adjusting the pH to 10 with NaOH. Then, 10 mL of AgNO3 0.1 M was added to the solution and the final mixture was heated for 30 min under controlled magnetic stirring. The contents of glucose and gelatin, as well as the temperature of synthesis varied according to the experimental design (Table 1). Before experimental analysis the colloidal solutions were stored at room temperature. Characterization of silver nanoparticles The UV–Vis spectra were obtained with a 1-cm path length quartz cell using a Cary 50 spectrophotometer (Varian, USA). The absorbance of the colloidal
Table 1 Experimental design Assay
Glucose (wt%)
Gelatin (wt%)
Temperature (°C)
1
0.54
0.36
65
2
0.54
0.36
85
3
0.54
0.72
65
4
0.54
0.72
85
5
0.90
0.36
65
6
0.90
0.36
85
7
0.90
0.72
65
8
0.90
0.72
85
9
1.02
0.64
75
10
0.72
0.84
75
11
0.72
0.54
92
12
0.41
0.54
75
13
0.72
0.23
75
14
0.72
0.54
58
15 16
0.72 0.72
0.54 0.54
75 75
17
0.72
0.54
75
18
0.72
0.54
75
19
0.72
0.54
75
20
0.72
0.54
75
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solutions was measured in the range of 300–800 nm. The size and shape of the nanoparticles were observed using a transmission electron microscopy (TEM, JEOL-JEM1010, Japan) operated at an accelerating voltage of 100 kV. A drop of silver nanoparticles solution was placed on a carbon-coated copper grid. The mean particle size was calculated by taking approximately 150 particles of each sample. The electron diffraction pattern of the nanoparticles was also obtained to confirm the crystalline structure of silver. Fourier transform infrared (FTIR) spectra of the precursors (AgNO3 and gelatin) and silver nanoparticles were recorded on a Spectrum One Spectrophotometer (Perkin Elmer, USA). For FTIR measurements of silver nanoparticles, a small amount of a colloidal solution of the particles was dried in a desiccator. The dried sample was mixed with KBr and the spectrum was obtained over a range of 4000–400 cm-1. Isolation of C. gloesporioides The phytopathogen C. gloesporioides was isolated from papaya fruits with symptoms of anthracnose. The infected part of the fruit was sanitized with alcohol 70%. Then, small portions were cut and immersed in a sodium hypochlorite solution (1%) for 1 min and washed with sterile distilled water. The portions of the fruit were placed on petri dishes containing the growth medium potato dextrose agar (PDA) and incubated at 27 ± 1 °C. Continuous reseeds of the mycelium on PDA were realized until obtain a pure culture. Conidia were observed with an optical microscopy and identification was according to a published description (Barnett and Hunter 1972). The identification of the fungus was done by Biol. David Bonilla Lo´pez (SENASICA, Me´xico DF). Antifungal activity of silver nanoparticles The antifungal activity of the silver nanoparticles was evaluated using the methodologies proposed by Bautista-Ban˜os et al. (2003) and Guo et al. (2007). Two different mean particle sizes (5 and 24 nm) and various concentrations (13, 26, and 52 lg silver/mL PDA) of silver nanoparticles in colloidal solution were added to sterilized PDA. Control dishes contained only PDA. The mycelium of C. gloesporioides was placed in the center of each petri dish and
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incubated at 27 ± 1 °C. Measurements of the colony diameter (cm) were carried out at 24 h intervals. The experiment concluded when the mycelium of fungi reached the edges of the control dish. The antifungal index (AI) was calculated at the end of the experiment by using the Eq. 1. D1 AI ð%Þ ¼ 1 100; ð1Þ D2 where D1 is the colony diameter in the test dishes and D2 is the colony diameter in the control dish. Statistical analysis In order to determine the effect of the synthesis variables on the size of the silver nanoparticles, the data of mean size were analyzed by the response surface methodology (RSM) with Design-Expert 7 statistical software (State-Ease Inc). There were five replicates for antifungal activity experiments and the results were evaluated by analysis of variance (ANOVA). Significant differences among mean values for mycelial growth were identified using Tukey’s studentized range test at P \ 0.05. Results and discussion Formation of silver nanoparticles After the addition of AgNO3 to the alkaline solution of glucose and gelatin, the color of the solution changed from colorless to brown indicating the formation of silver nanoparticles. The possible reaction between glucose and silver ion in gelatin solution can be written as follows: 2Agþ þ 2OH ! Ag2 O þ H2 O; ð2Þ Ag2 O þ CH2 OH ðCHOHÞ4 CHO þ 2 Gelatin ! CH2 OH ðCHOHÞ4 COOH þ 2AgðGelatinÞ # : ð3Þ According to Eq. 2, silver ions in aqueous solution react with hydroxyl ions forming silver oxide. Subsequently, the silver oxide is reduced by glucose generating silver nanoparticles (Eq. 3). The function of the gelatin is to form a protective layer on the surface of nanoparticles with the aim of preventing their aggregation and thus maintain the stability in colloidal solution.
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UV–Vis spectroscopy
2.1
TEM characterization The TEM images confirmed the presence of spherical silver nanoparticles with a relatively small size (Fig. 3). According to Fig. 3a, the synthesis conditions of the assay 10, favored the production of silver 2.1
Absorbance (a. u.)
1.8 1.5
Assay 11 Assay 9 Assay 20 Assay 2 Assay 1
1.2 0.9 0.6 0.3 0.0 300
400
500
600
700
800
Wavelength (nm) Fig. 1 UV–Vis absorption spectra of silver nanoparticles showing a single symmetric absorption band
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30 min 3 months
1.8
Absorbance (a. u.)
Figure 1 presents some UV–Vis absorption spectra of the colloidal solutions of silver nanoparticles obtained in this work at different conditions of synthesis (Table 1). All the UV–Vis spectra presented only a single symmetric absorption band, in the wavelength interval characteristic of silver nanoparticles. The position and shape of the surface plasmon absorption band are dependent on the size, shape, and polydispersity of the particles (Mitra and Bhaumik 2007; Slistan-Grijalva et al. 2008). If the size of the particle increases, the absorption band tends to shift to longer wavelength. Also the number of absorption peaks increases as the symmetry of the nanoparticle decreases (Pal et al. 2007). Then, based on the position, shape, and width of the absorption bands, it was possible to estimate the predominant presence of spherical silver nanoparticles with a size relatively small (\25 nm). Even after 3 months, the characteristics of the absorption bands remained without significant changes (Fig. 2), confirming the colloidal stability. These results were later confirmed by TEM.
1.5 1.2 0.9 0.6 0.3 0.0 300
400
500
600
700
800
Wavelength (nm)
Fig. 2 UV–Vis absorption spectra of silver nanoparticles obtained under conditions of assay 16. The spectra were recorded immediately after synthesis and after 3 months
nanoparticles with a mean particle size of 24 ± 6.9 nm; meanwhile working with the conditions of the assay 6, the mean size was reduced to 5 ± 2.4 nm (Fig. 3b). Particle size distributions were determined by measuring the diameter of more than 150 particles on the TEM images. Figure 4a, b show the corresponding histograms obtained from Fig. 3a, b, respectively. By electron diffraction pattern (Fig. 5) it was possible to confirm the crystalline nature of the silver nanoparticles. The interplanar spacing was determined from the rings of the diffraction pattern and indexed according to JCPDS card No. 04-0783, showing a structure face-centered cubic (fcc). The values of average size were analyzed by means of the RSM (Fig. 6). In accordance with Fig. 6a (58 °C), small nanoparticles (*3.5 nm) could be obtained using the lowest gelatin content (0.23 wt%) and the lowest glucose concentration (0.41 wt%). On the other hand, nanoparticles with a bigger size (*29 nm) could be synthesized under a high concentration of protein (0.84 wt%) and the lowest concentration of reducing agent (0.41 wt%). When the temperature is increased until 92 °C, the effects of gelatin and glucose concentrations on the size of the silver nanoparticles were opposite to that observed at 58 °C (Fig. 6b). The size of metal nanoparticles is greatly influenced by the concentration of reducing agent. In general, an increase in the concentration of reducing agent increases the reduction rate of metal ions, leading to the formation of smaller metal
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Fig. 3 TEM micrographs silver nanoparticles obtained under conditions of assay 10 (a) and assay 6 (b)
Fig. 4 Particle size distribution histograms of silver nanoparticles shown in Fig. 3a and b
12
(a)
25 20
Frequency
Frequency
10
(b)
8 6 4
15 10 5
2
0
0 10
20
30
40
Particle size (nm)
50
3
6
9
12
Particle size (nm)
Fig. 5 Electron diffraction pattern of silver nanoparticles (assay 10)
nanoparticles (Tan et al. 2004). In accordance with Sun and Luo (2005), when the concentration of capping agent is increased it is possible to obtain a particle with
a smaller size. However, in this investigation, this fact only was observed using a temperature of synthesis of 92 °C. It was possible to observe that the temperature
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(b)
29.0
40.0
22.5
31.5
Particle size (nm)
Particle size (nm)
(a)
16.0 9.5
23.0 14.5 6.0
3.0
0.84
1.02
0.56
0.38 0.23
0.87
0.69 0.72
0.53
0.72
0.53
Gelatin (wt%)
1.02
0.84
0.87
0.69
Glucose (wt%)
Gelatin (wt%)
0.56
0.38 0.23
0.41
Glucose (wt%)
0.41
of synthesis favored importantly the growth of the particles. Similar results were reported by Luo et al. (2005). Equation 4 represents the mathematical model for the particle size, presenting a moderate fitting with the experimental data (R2 [ 60%). Particle size ¼ 53:18 39:87 ðGlucoseÞ þ 78:36 ðGelatinÞ 1:63 ð TÞ
% Transmittance
Fig. 6 Response surface plots of the combined effects of gelatin and glucose contents on the particle size at 58 °C (a) and 92 °C (b)
(a) (b)
1376
(c)
1640 1540 1385
3400
þ 25:48 ðGlucoseÞ2
1648 3417
þ 55:49 ðGelatinÞ2 þ 0:011 ð TÞ2 94:71 ðGlucoseÞ ðGelatinÞ þ 0:80 ðGlucoseÞð TÞ 0:98 ðGelatinÞð TÞ:
ð4Þ
4000
1240 1548
3000
2000
1000 -1
Wavenumber (cm )
FTIR spectroscopy
Fig. 7 FTIR spectra of: AgNO3 (a), silver nanoparticles (assay 6) (b) and gelatin (c)
The main utility of the FTIR spectroscopy in the characterization of metal nanoparticles is to detect chemical species that interact with the surface of the particles (Baker et al. 2004). Figure 7 shows the FTIR spectra of the precursors (AgNO3 and gelatin) and silver nanoparticles. The spectrum of the gelatin revealed characteristic absorption bands at 3417 cm-1 (amide A, N–H stretching and O–H bending and stretching), 1648 cm-1 (amide I, C=O stretching), 1535 cm-1 (amide II, N–H bending and C–N stretching) and 1240 cm-1 (amide III, N–H bending). The spectrum of the AgNO3 displayed a very intense absorption band at 1376 cm-1, which is characteristic of the ion pair, Ag?NO3-. On the other
hand, in the spectrum of the silver nanoparticles, the absorption bands corresponding to the amide groups, shifted to lower wavenumber values. These changes on the position bands may be due to the interaction with silver nanoparticles. Also the band intensity of the ion pair Ag?NO3- decreased and shifted to a higher wavenumber value (1385 cm-1). This peak, centered at 1385 cm-1, is characteristic of the NO3- ion in free form, and the absorption band displacement is caused by a change in the electronic environment of the anion, as a result of the separation of its counterpart Ag? (Cho and So 2006). With these results it is possible to deduce that silver nanoparticles interacted with the different
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amide and hydroxyl groups of gelatin, assuming that these interactions are responsible in a great manner of the stability of colloidal solutions. According to Basavaraja et al. (2008) the carbonyl groups of amino acids and proteins have the ability to link metal particles and thus prevent their agglomeration.
Table 2 Antifungal index of silver nanoparticles against C. gloesporioides Treatment (lg silver/mL PDA)
Figure 8 shows the effect of the size and concentration of silver nanoparticles in colloidal solution, on the mycelial growth of C. gloesporioides. It is evident that the addition of silver nanoparticles to PDA medium caused an important decrease in the growth rate of the fungus. In accordance with the ANOVA, differences among the treatments and control were statistically significant (P \ 0.001). Also the reduction in the mycelial growth was higher as the concentration of silver increased in PDA medium. However, differences were not observed (P [ 0.05) for the effect of the mean particle size (5 and 24 nm) on the reduction of the mycelial growth. The last fact could be explained by the short difference (19 nm) between the two mean particle sizes of silver nanoparticles. According to Jo et al. (2009), the mechanism of antifungal activity of silver nanoparticles is based on the possibility that nanoparticles may attach to and penetrate the cell membrane and kill spores. Table 2 shows the antifungal index for each treatment at the end of the experiment (7 days). It was possible to observe that using the highest concentration of silver nanoparticles (56 lg silver/ mL PDA), the inhibition of the fungus reached almost 90%. Then, at the concentrations proposed in this study, the silver nanoparticles had a fungistatic effect
13
73 ± 3.4
74 ± 2.5
82 ± 3.9
82 ± 5.1
56
89 ± 3.1
89 ± 3.2
on C. gloesporioides. Petica et al. (2008) also reported a fungistatic activity of silver nanoparticles on fungus species as Aspergillus, Penicillium, and Trichoderma. On the other hand, Kim et al. (2009) reported that the decrease in the growth of fungus Raffaelea sp. was in function on the concentration of silver nanoparticles.
Conclusions Spherical and relatively small silver nanoparticles were prepared by reducing AgNO3 with glucose in the presence of gelatin as a protective agent. The mean particle size ranged between 5 and 24 nm and was a function of the synthesis conditions. The colloidal solutions remained stable at room temperature for more than 3 months. The silver nanoparticles presented a dose-dependent fungistatic activity on C. gloesporioides. The inhibition of the fungus reached almost 90% with a low silver nanoparticles concentration (56 lg silver/mL PDA). Further research should focus in the application of silver nanoparticles on horticultural products to control postharvest diseases.
(a)
10 13 µg silver/mL 26 µg silver/mL 56 µg silver/mL Control
8
24
26
Mycelial growth (cm)
Mycelial growth (cm)
10
Average particle size (nm) 5
Evaluation of antifungal activity of silver nanoparticles
Fig. 8 Effect of silver nanoparticles at different concentrations on mycelial growth of C. gloesporioides during 7-days incubation period: 5 nm mean particle size (a) and 25 nm mean particle size (b)
Antifungal index (%)
6 4 2 0
(b) 13 µg silver/mL 26 µg silver/mL 56 µg silver/mL Control
8 6 4 2 0
2
3
4
5
Time (d)
6
7
2
3
4
5
6
7
Time (d)
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2532 Acknowledgments This work was financially supported by Consejo Nacional de Ciencia y Tecnologı´a (CONACYT) through project no. 90019 and SIP project no. 20082511. The authors would like to thank Dr. Geonel Gattorno for the technical assistance in electron diffraction and FTIR.
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