Materials Science and Engineering C 41 (2014) 249–254
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of poly acrylic acid modified silver nanoparticles and their antimicrobial activities Zhihui Ni a, Zhihua Wang b, Lei Sun a,⁎, Binjie Li c, Yanbao Zhao a a b c
Key Laboratory for Special Functional Materials of Ministry of Education, Henan University, Kaifeng 475004, PR China Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 17 March 2014 Accepted 22 April 2014 Available online 2 May 2014 Keywords: Synthesis Poly acrylic acid Silver nanoparticles Antimicrobial property
a b s t r a c t Poly acrylic acid modified silver (Ag/PAA) nanoparticles (NPs) have been successfully synthesized in the aqueous solution by using tannic acid as a reductant. The structure, morphology and composition of Ag/PAA NPs were characterized by various techniques such as X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible absorption spectroscopy (UV–vis) and thermogravimetry analysis (TGA). The results show that PAA/Ag NPs have a quasi-ball shape with an average diameter of 10 nm and exhibit well crystalline, and the reaction conditions have some effect on products morphology and size distribution. In addition, the as-synthesized Ag/PAA NPs antimicrobial activities against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) were evaluated by the methods of broth dilution, cup diffusion, optical density (OD600) and electron microscopy observation. The as-synthesized Ag/PAA NPs exhibit excellent antibacterial activity. The antimicrobial mechanism may be attributed to the damaging of bacterial cell membrane and causing leakage of cytoplasm. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Microbial threats on human health and safety have become a serious public concern recently. To prevent disease, a great effort has been devoted to the development of new and effective antimicrobial materials [1–3]. Antibacterial materials are mainly divided into inorganic, organic, and natural materials. The natural antibacterial materials present usually a narrow application scope, and the organic antibacterial materials with poor heat resistance are easy to cause drug resistance of bacteria. Inorganic antibacterial materials exhibit better performance than organic antibacterial materials in durability, heat resistance, and low occurrence of antibiotic resistance for bacterial strains. So it has attracted increasing attention in recent years [4,5]. For example, Ag+ ions are broad-spectrum biocide and can effectively inhibit the growth of bacteria, fungi and algae [6,7]. Many inorganic nanoparticles (NPs) have shown severe cytotoxicity, which open the possibility of formulation of a new generation of bactericidal materials [8–11]. In particular, silver NPs have been extensively studied due to their high antibacterial activity with minimal perturbation to human cells [12]. For example, silicon nanowires decorated with silver NPs show efficient antibacterial activity [13]. However, most studies were focused on the antibacterial performance and
mechanisms of Ag NPs [14,15], few effort was related to the development of environmental friendly and biocompatibility preparing method. In this paper, we present a convenient one-pot procedure for the preparation of highly water-soluble poly acrylic acid modified silver (Ag/PAA) NPs using tannic acid as reductant. PAA molecules are in rich of carboxyl groups on both sides of polymer chains, which can bind the polymer on the surface of Ag NPs and make the modified nanoparticles water-soluble and stable [16–18]. Characterizations of the as-synthesized Ag/PAA NPs were achieved by various techniques such as X-ray powder diffraction (XRD), transmission electron microscopy (TEM), ultraviolet–visible absorption spectroscopy (UV– vis), Fourier transformation infrared spectrometry (FTIR), and thermogravimetry analysis (TGA). The antibacterial activity of Ag/PAA NPs against Gram-negative bacteria of Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Gram-positive bacteria of Staphylococcus aureus (S. aureus) was evaluated by the methods of broth dilution, cup diffusion, optical density (OD600) and scanning electron microscopy (SEM). 2. Experimental 2.1. Reagents
⁎ Corresponding author. Tel.: +86 371 23881358. E-mail address: [email protected] (L. Sun).
http://dx.doi.org/10.1016/j.msec.2014.04.059 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Silver nitrate (AgNO3), PAA (Mw = 800–1000) and acetone were purchased from Chemical Reagent Corporation of Chinese National
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Medical Group (Beijing, China). Tannic acid, ammonia (NH3·H2O, 25%– 28%) and sodium chloride (NaCl) were purchased from Tianjin Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). All of the above‐mentioned reagents were analytical grade (AR) and used as received without further purification. Nutrient agar and broth medium were both biochemical reagents (BR) and purchased from Beijing Aoboxing Biotechnology Corporation (Beijing, China). E. coli, P. aeruginosa and S. aureus bacterial strains were purchased from China General Microbiological Collection Center (CGMCC). Distilled water was used throughout the experiment.
minimum bactericidal concentration (MBC) were used to assess the antimicrobial activities of the hybrid NPs, which were determined by the two-fold serial broth dilution method [20]. For MIC test, the serial broth solutions with different concentrations (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 μg/mL) of the Ag/PAA NPs were mixed with 20 μL bacterial suspension at a concentration of 108 colonyforming units (CFU)/mL, and incubated at 37 °C for 22 h. The Ag/PAA
2.2. Instrumentation and characterization XRD patterns were collected on an X’pert Philips diffractometer equipped with Cu Kα radiation (λ = 1.5418 Å) and operating at 40 kV and 40 mA. SEM images were collected using a JEOL JSM5600LV scanning electron microscope at an acceleration voltage of 20 kV. TEM images were obtained using a JEOL JEM-100CX transmission electron microscope. The samples were prepared by placing a drop of primary sample on a copper grid. FTIR spectra were taken on an AVATAR 360 Fourier transform infrared spectrometer. UV–vis absorption spectra were recorded using a UNICORN 540 UV–vis absorption spectrophotometer. TGA was conducted in nitrogen on a Seiko EXSTAR 6000 thermal analyzer at a scanning rate of 10 °C/min. 2.3. Preparation of Ag/PAA NPs In a typical procedure, 60 mL of PAA (0.04 mol/L) and 40 mL of AgNO3 (0.12 mol/L) solution were injected into a 500 mL flask, respectively. The solution was stirred and heated to 50 °C for 0.5 h. Then, 130 mL tannic acid (0.006 mol/L) solution was added into the flask. When the color of the solution changed from colorless to light yellow, 12 mL NH3·H2O (0.5 mol/L) was added, and the color of the solution became rufous immediately. Then the particles were separated by centrifugation at 8500 r/min for 15 min and washed with acetone and distilled water several times. At last, the Ag/PAA NPs were obtained by vacuum drying at ambient temperature. A series of PAA modified Ag nanoparticles with different molar ratio (namely, 1:4, 1:2, 1:1) of PAA to Ag was obtained through changing the added amount of PAA but fixing that of AgNO3 and tannic acid in the experiments. 2.4. Antimicrobial activity testing In order to evaluate the antibacterial activity, Ag/PAA NPs, E. coli, P. aeruginosa and S. aureus were selected as indicators. Glassware, suction nozzles and culture medium were sterilized in an autoclave at a high pressure of 0.1 MPa and a temperature of 121 °C for 20 min before experiments [19]. The minimal inhibitory concentration (MIC) and the
Fig. 1. XRD patterns of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Fig. 2. TEM images of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1, the insets are histograms of the particles size distribution.
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Fig. 3. The UV–vis spectra of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Fig. 5. TG curves of (a) Ag/PAA NPs with the molar ratio of PAA/Ag at 1:2 and (b) pure Ag particles without surface modification.
NPs-free bacterial broth suspension was made as the control. The MIC is defined as the lowest concentration of samples that inhibited visible growth of bacteria by turbid-metric method after incubation. The MBC is the minimum concentration of the sample required to kill 99.9% bacteria after a defined period of incubation. In the test, the bacterial suspension with different amount of Ag/PAA NPs was coated on the nutrient agar plate. Subsequently, the agar plates with bacterial suspension were incubated at 37 °C for 24 h. The number of survival colonies was counted to get the MBC of the hybrid Ag/PAA NPs. The antibacterial activities were also performed by cup diffusion method. Firstly, 100 μL bacteria suspensions were put uniformly on the surface of the agar Petri dish and solidified. Secondly, the sterile Oxford cups with a diameter of 6 mm were placed on the Petri dish and marked. Thirdly, the sample solutions with different concentrations were added into the Oxford cups. At the same time, the normal saline as control sample was dropped into the central cups. Finally, the agar Petri dishes were placed in the constant temperature incubator at 37 °C for 22 h. The antibacterial effect was evaluated by the diameter of visible transparent inhibitory zone. The antibacterial activity was also studied by the bacterial growth kinetics in broth media [21]. 2.5 mL of sample solutions with different concentrations (2, 40, 100 μg/mL) was mixed with 2.5 mL of sterilized broth media in 10 mL culture tubes. Then, 50 μL of bacterial suspensions was added into these tubes, and they were incubated at 37 °C for 48 h. These tubes were withdrawn at set time intervals to measure the optical density (OD) at 600 nm by using the UV–vis absorption spectrophotometer. As the control group, 50 μL of bacterial suspension was injected into 5 mL broth medium and performed to the same procedure as the
above. Finally, the OD values were plotted against the time to obtain the growth curve. In addition, the change of morphology of bacteria treated with Ag/PAA NPs was evaluated by SEM and TEM [22]. 2 mL of broth medium and 40 μL of bacterial suspensions were mixed and cultured at 37 °C for 6 h. Then, 100 μg/mL Ag/PAA NPs was added and allowed to culture at the same condition for another 6 h. At last, the bacteria were collected by centrifugation. To fabricate bacterial SEM and TEM samples, the bacteria were fixed with a diluted glutaraldehyde solution (2.5%) at −4 °C for 30 min and centrifuged at 6000 r/min for 5 min. After dehydration with series of alcohol solution, the collected bacteria were observed by SEM and TEM. 3. Results and discussion 3.1. XRD patterns of Ag/PAA NPs Fig. 1 shows that XRD patterns of the as-synthesized Ag/PAA NPs with different molar ratio of PAA/Ag. It is clear to see that the three samples present multiple diffraction peaks, and the peaks at 2θ of 38.1°, 44.3°, 64.4°, 77.4° and 81.5° are assigned to diffractions from the (111), (200), (220), (311) and (222) lattice planes of face-centered cubic (fcc) of silver (JCPDS No.04-0783), respectively. There are no diffraction peaks of Ag2O, which suggests that the existence of modification layer of PAA can prevent the oxidation of Ag NPs. 3.2. Morphologies of Ag/PAA NPs Fig. 2 shows the TEM images of the as-synthesized Ag/PAA NPs with different molar ratio of PAA/Ag, the insets are histograms of the particles size distribution. The histograms were measured and calculated from TEM images through size statistics software of scion image [23]. It can be seen from Fig. 2(b) when the molar ratio of PAA/Ag is 1:2, the sample exhibits uniform sphere shape, and have an average size of 8.5 nm with a size distribution of ±1.8 nm. While the molar ratio of PAA/Ag is 1:4, the obtained sample (Fig. 2(a)) is quasi sphere in shape. The average particles size and distribution is 8.8 and ±2.2 nm, respectively. When Table 1 MIC and MBC numerical values of Ag, Ag/PAA NPs and PAA. MIC (μg/mL)
Fig. 4. FTIR spectra of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Ag PAA/Ag PAA
MBC (μg/mL)
E. coli
P. aeruginosa
S. aureus
E. coli
P. aeruginosa
S. aureus
125 3.9 ——
250 7.8 1000
125 3.9 1000
250 15.6 ——
500 62.5 ——
250 15.6 ——
——: indicating no antibacterial properties.
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Fig. 6. Inhibition zone photographs of Ag/PAA NPs against bacteria E. coli (a, b), P. aeruginosa (c, d) and S. aureus (e, f). (1–8) Samples correspond concentrations with 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL and the control group in the middle is 9% normal saline (NS).
the molar ratio of PAA/Ag increased to 1:1, obvious aggregated particles emerged, the mean particles size of 10.0 nm was estimated, and the particles size distribution of ± 2.2 nm was calculated. From TEM
observation, it can be concluded that the optimum molar ratio of PAA/Ag is 1:2, so this sample is chosen for thermal analysis and antibacterial test. 3.3. Optical properties of Ag/PAA NPs Fig. 3 shows UV–vis absorption spectra of the Ag/PAA NPs with different molar ratio of PAA/Ag. It can be seen that the appearance of peaks around 424 nm of three curves clearly indicates the formation of Ag NPs, which was attributed to the excitation of surface plasmon resonance (SPR) [24,25] or inter-band transition of silver NPs. The obtuse absorption peak in Fig. 3(a) is possibly assigned to little PAA coated on the surface and the wide particles size distribution of Ag/PAA NPs. It also can be found from Fig. 3(b) compared with Fig. 3(a) that a blue shift of the in-plane dipole resonance from 426 to 424 nm occurs, which suggests that the particles obtained here are truncated. 3.4. FTIR spectra of Ag/PAA NPs
Fig. 7. Growth curves of E. coli (A) and S. aureus (B) with different concentration Ag/PAA NPs treated (b, c, d) and bacterial without Ag/PAA as control test (a).
To gain further insight into the interaction of PAA with the Ag NPs surface, FTIR spectroscopy is used to analyze the organic functional groups. Fig. 4 shows FTIR spectra of Ag/PAA NPs with different molar ratio of PAA/Ag. As is shown in Fig. 4(b), a wide band at 3426 cm−1 is attributed to the stretching vibration of hydroxyl group in the adsorbed water. The band at 2927 cm−1 is assigned to the stretching vibration of –CH2 group from the long aliphatic chains. The band at 1630 cm−1 is assigned to stretching vibration of the free carbonyl groups. The bands at 1390 cm− 1 and 1260 cm− 1 can be assigned to the symmetric and anti-symmetric vibration modes of –COO− groups, indicating the adsorption of PAA on the nanoparticles through the bidentate bonds [26–28]. The wide band at 814 cm − 1 is the skeleton vibration of –C–C–. Compared with the relative intensity of each absorption bands at 1390, 1260 and 814 cm−1 in Fig. 4(a–c), it can be seen that the relative intensity gradually increased with the augment of molar ratio of PAA/Ag. The band at 679 cm−1 is assigned to stretching vibration of O-Ag [29,30], indicating that a coordination reaction between –COO and Ag atoms may occur.
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Fig. 8. SEM images of the normal and treated bacteria with 100 μg/mL of Ag/PAA NPs solution for E. coli (a, b), P. aeruginosa (c, d), and S. aureus (e, f).
3.5. Thermal analysis of Ag/PAA NPs Fig. 5 shows TG curves of the as-prepared PAA/Ag NPs with the molar ratio of PAA/Ag at 1:2 (a) and pure Ag particles without surface modification (b). It can be seen from Fig. 5(a) that Ag/PAA NPs exhibit obviously two weight loss processes. The first weight loss process (about 3%) below 100 °C may be attributed to the release of adsorbed water. The second mass loss process (about 15%) in the range of 160 °C to 560 °C is attributed to the decomposition of PAA binding on the nanoparticles. By comparison, for pure Ag NPs, there is almost no mass loss in the whole temperature raising range from ambient temperature to 800 °C. Accordingly, it can be concluded that PAA has been coated on the surfaces of Ag NPs through chemical reaction, which is consistent with the FTIR analysis. 3.6. Antimicrobial activities of Ag/PAA NP Table 1 shows the MIC and MBC values of Ag, Ag/PAA NPs, PAA against E. coli, S. aureus and P. aeruginosa measured by broth dilution method. The average particle size of Ag/PAA NPs is 8.5 nm (the sample with PAA/Ag molar ratio of 1:2), yet that of Ag NPs without PAA is about
Fig. 9. TEM images of the normal bacteria (a) and treated E. coli with 100 μg/mL of Ag/PAA NPs (b), the insets are partial enlarged images.
400 nm and the nanoparticles have severe aggregation. From Table 1, it can be seen that the MIC and MBC value of Ag against E. coli, S. aureus and P. aeruginosa is more than 125 μg/mL. PAA almost exhibits no antibacterial property. However, the MIC and MBC values of Ag/PAA NPs against E. coli, S. aureus and P. aeruginosa are lower than 7.8 and 62.5 μg/mL, respectively, which are obviously better than those of Ag and PAA. These results show that the Ag/PAA NPs have excellent antibacterial properties. From Table 1, it also can be seen that Ag/PAA NPs exhibit equal antibacterial against Gram-negative bacteria of E. coli and Gram-positive bacteria of S. aureus, which is some different from the reference [31]. This may be attributed to the Ag NPs size and stable agents are different in the two work. Fig. 6 shows inhibition zone photographs of Ag/PAA NPs against E. coli (a, b), P. aeruginosa (c, d), S. aureus (e, f), respectively. Samples (1–8) corresponds to the concentrations with 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL and the control group in the middle was 9% normal saline (NS) [19,32]. Measurement results show that the diameter of inhibition zone of Ag/PAA NPs against E. coli, P. aeruginosa and S. aureus are 10.0, 16.0 and 20.0 mm at the sample concentration 125 μg/mL, respectively. There is no obvious inhibition zone for the control test of NS. Besides the MIC measurement, the inhibition zone test results further indicate intuitively that the as-synthesized Ag/PAA NPs exhibit bacterial inhibition effects. The antibacterial properties were also investigated by testing the bacterial growth curves in liquid broth media. The time-dependent changes of bacterial growth were monitored by measuring OD600. Fig. 7 shows the growth curves of typical Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus) with different concentration Ag/PAA NPS for 48 h, the control test is bacteria in broth medium without Ag/PAA NPs. It is clearly to see that the sample at all tested concentrations has strong suppression of proliferation of tested strains. For E. coli, when the concentration was over 20 μg/mL, the sample could inhibit completely the growth of E. coli during the whole 48 h (Fig. 7(a)). However, when the concentration was below MIC (3.9 μg/mL), it was not enough to inhibit the growth of E. coli within 48 h, and there was a growth delay of E. coli. For S. aureus, the solution of 20 μg/mL and 60 μg/mL could completely inhibit the growth of bacteria. Similarly, the growth curves of S. aureus with the concentration 2 μg/mL of
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Ag/PAA NPs also showed a lag phase compared to the control test of S. aureus (Fig. 7(b)). The influence of Ag/PAA solution to bacteria was further evaluated by SEM through observing the change of morphology of the original and treated bacteria. Fig. 8 shows SEM images of the normal and treated bacteria with 100 μg/mL of Ag/PAA NPs solution for E. coli (a, b), P. aeruginosa (c, d), and S. aureus (e, f). It can be seen from Fig. 8(a) that original E. coli exhibit uniform short rod morphologies with smooth surfaces while as shown in Fig. 8(b), the treated E. coli cells exhibit significantly changes in reduced length and rough surfaces, which indicates the occurrence of interaction of Ag/PAA NPs and the distinct damage of bacterial cells. The similar phenomenon can be observed by comparison of Fig. 8(d) and (e) of untreated and treated bacteria of P. aeruginosa. The normal S. aureus cells observed from Fig. 8(e) is spherical in shape with an average diameter of 1 μm and has smooth cell surfaces, whereas the membrane deformation and rough surfaces formation can be seen along with the appearance of cell debris in the case of treated cells as shown in Fig. 8(f). In order to demonstrate further the integration of Ag/PAA NPs and cell wall, E. coli was chosen as a typical bacterium for TEM observation. Fig. 9 shows TEM images of the normal E. coli (a) and treated E. coli with 100 μg/mL of Ag/PAA NPs (b), the insets are partial enlarged images. It is obvious to see from Fig. 9 that after treated with Ag/PAA NPs, the rodlike cell of E. coli broke into fragments, and Ag/PAA NPs were attached on the surfaces of the bacterial cell. Although the exact action mechanism of Ag NPs against bacteria is not very well known, the possible antibacterial action model can be proposed based the above‐mentioned results. Ag/PAA NPs can attach to the surface of the cell membrane and disturb its functions, and then it penetrates the bacteria to cause further damage, such as leakage of cytoplasm. In addition, Ag/PAA NPs may release Ag+, which make an additional contribution to the bactericidal effect [33,34]. 4. Conclusions Ag NPs modified by PAA is successfully synthesized by chemical reduction method. The particles’ morphologies and sizes were depended strongly on the molar ratio of surfactant agent to Ag. The Ag/PAA NPs have excellent water solubility, stability and biological compatibility. Ag/PAA NPs exhibit effective bacteriostasis and bactericidal activity against E. coli, S. aureus and P. aeruginosa. The possible antibacterial mechanism against bacteria is proposed. Acknowledgements The authors are grateful for the financial support provided by National Natural Science Foundation of China (50701016, 21271062), China
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of poly acrylic acid modified silver nanoparticles and their antimicrobial activities Zhihui Ni a, Zhihua Wang b, Lei Sun a,⁎, Binjie Li c, Yanbao Zhao a a b c
Key Laboratory for Special Functional Materials of Ministry of Education, Henan University, Kaifeng 475004, PR China Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 17 March 2014 Accepted 22 April 2014 Available online 2 May 2014 Keywords: Synthesis Poly acrylic acid Silver nanoparticles Antimicrobial property
a b s t r a c t Poly acrylic acid modified silver (Ag/PAA) nanoparticles (NPs) have been successfully synthesized in the aqueous solution by using tannic acid as a reductant. The structure, morphology and composition of Ag/PAA NPs were characterized by various techniques such as X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible absorption spectroscopy (UV–vis) and thermogravimetry analysis (TGA). The results show that PAA/Ag NPs have a quasi-ball shape with an average diameter of 10 nm and exhibit well crystalline, and the reaction conditions have some effect on products morphology and size distribution. In addition, the as-synthesized Ag/PAA NPs antimicrobial activities against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) were evaluated by the methods of broth dilution, cup diffusion, optical density (OD600) and electron microscopy observation. The as-synthesized Ag/PAA NPs exhibit excellent antibacterial activity. The antimicrobial mechanism may be attributed to the damaging of bacterial cell membrane and causing leakage of cytoplasm. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Microbial threats on human health and safety have become a serious public concern recently. To prevent disease, a great effort has been devoted to the development of new and effective antimicrobial materials [1–3]. Antibacterial materials are mainly divided into inorganic, organic, and natural materials. The natural antibacterial materials present usually a narrow application scope, and the organic antibacterial materials with poor heat resistance are easy to cause drug resistance of bacteria. Inorganic antibacterial materials exhibit better performance than organic antibacterial materials in durability, heat resistance, and low occurrence of antibiotic resistance for bacterial strains. So it has attracted increasing attention in recent years [4,5]. For example, Ag+ ions are broad-spectrum biocide and can effectively inhibit the growth of bacteria, fungi and algae [6,7]. Many inorganic nanoparticles (NPs) have shown severe cytotoxicity, which open the possibility of formulation of a new generation of bactericidal materials [8–11]. In particular, silver NPs have been extensively studied due to their high antibacterial activity with minimal perturbation to human cells [12]. For example, silicon nanowires decorated with silver NPs show efficient antibacterial activity [13]. However, most studies were focused on the antibacterial performance and
mechanisms of Ag NPs [14,15], few effort was related to the development of environmental friendly and biocompatibility preparing method. In this paper, we present a convenient one-pot procedure for the preparation of highly water-soluble poly acrylic acid modified silver (Ag/PAA) NPs using tannic acid as reductant. PAA molecules are in rich of carboxyl groups on both sides of polymer chains, which can bind the polymer on the surface of Ag NPs and make the modified nanoparticles water-soluble and stable [16–18]. Characterizations of the as-synthesized Ag/PAA NPs were achieved by various techniques such as X-ray powder diffraction (XRD), transmission electron microscopy (TEM), ultraviolet–visible absorption spectroscopy (UV– vis), Fourier transformation infrared spectrometry (FTIR), and thermogravimetry analysis (TGA). The antibacterial activity of Ag/PAA NPs against Gram-negative bacteria of Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Gram-positive bacteria of Staphylococcus aureus (S. aureus) was evaluated by the methods of broth dilution, cup diffusion, optical density (OD600) and scanning electron microscopy (SEM). 2. Experimental 2.1. Reagents
⁎ Corresponding author. Tel.: +86 371 23881358. E-mail address: [email protected] (L. Sun).
http://dx.doi.org/10.1016/j.msec.2014.04.059 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Silver nitrate (AgNO3), PAA (Mw = 800–1000) and acetone were purchased from Chemical Reagent Corporation of Chinese National
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Medical Group (Beijing, China). Tannic acid, ammonia (NH3·H2O, 25%– 28%) and sodium chloride (NaCl) were purchased from Tianjin Kemiou Chemical Reagent Co. Ltd. (Tianjin, China). All of the above‐mentioned reagents were analytical grade (AR) and used as received without further purification. Nutrient agar and broth medium were both biochemical reagents (BR) and purchased from Beijing Aoboxing Biotechnology Corporation (Beijing, China). E. coli, P. aeruginosa and S. aureus bacterial strains were purchased from China General Microbiological Collection Center (CGMCC). Distilled water was used throughout the experiment.
minimum bactericidal concentration (MBC) were used to assess the antimicrobial activities of the hybrid NPs, which were determined by the two-fold serial broth dilution method [20]. For MIC test, the serial broth solutions with different concentrations (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 μg/mL) of the Ag/PAA NPs were mixed with 20 μL bacterial suspension at a concentration of 108 colonyforming units (CFU)/mL, and incubated at 37 °C for 22 h. The Ag/PAA
2.2. Instrumentation and characterization XRD patterns were collected on an X’pert Philips diffractometer equipped with Cu Kα radiation (λ = 1.5418 Å) and operating at 40 kV and 40 mA. SEM images were collected using a JEOL JSM5600LV scanning electron microscope at an acceleration voltage of 20 kV. TEM images were obtained using a JEOL JEM-100CX transmission electron microscope. The samples were prepared by placing a drop of primary sample on a copper grid. FTIR spectra were taken on an AVATAR 360 Fourier transform infrared spectrometer. UV–vis absorption spectra were recorded using a UNICORN 540 UV–vis absorption spectrophotometer. TGA was conducted in nitrogen on a Seiko EXSTAR 6000 thermal analyzer at a scanning rate of 10 °C/min. 2.3. Preparation of Ag/PAA NPs In a typical procedure, 60 mL of PAA (0.04 mol/L) and 40 mL of AgNO3 (0.12 mol/L) solution were injected into a 500 mL flask, respectively. The solution was stirred and heated to 50 °C for 0.5 h. Then, 130 mL tannic acid (0.006 mol/L) solution was added into the flask. When the color of the solution changed from colorless to light yellow, 12 mL NH3·H2O (0.5 mol/L) was added, and the color of the solution became rufous immediately. Then the particles were separated by centrifugation at 8500 r/min for 15 min and washed with acetone and distilled water several times. At last, the Ag/PAA NPs were obtained by vacuum drying at ambient temperature. A series of PAA modified Ag nanoparticles with different molar ratio (namely, 1:4, 1:2, 1:1) of PAA to Ag was obtained through changing the added amount of PAA but fixing that of AgNO3 and tannic acid in the experiments. 2.4. Antimicrobial activity testing In order to evaluate the antibacterial activity, Ag/PAA NPs, E. coli, P. aeruginosa and S. aureus were selected as indicators. Glassware, suction nozzles and culture medium were sterilized in an autoclave at a high pressure of 0.1 MPa and a temperature of 121 °C for 20 min before experiments [19]. The minimal inhibitory concentration (MIC) and the
Fig. 1. XRD patterns of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Fig. 2. TEM images of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1, the insets are histograms of the particles size distribution.
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Fig. 3. The UV–vis spectra of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Fig. 5. TG curves of (a) Ag/PAA NPs with the molar ratio of PAA/Ag at 1:2 and (b) pure Ag particles without surface modification.
NPs-free bacterial broth suspension was made as the control. The MIC is defined as the lowest concentration of samples that inhibited visible growth of bacteria by turbid-metric method after incubation. The MBC is the minimum concentration of the sample required to kill 99.9% bacteria after a defined period of incubation. In the test, the bacterial suspension with different amount of Ag/PAA NPs was coated on the nutrient agar plate. Subsequently, the agar plates with bacterial suspension were incubated at 37 °C for 24 h. The number of survival colonies was counted to get the MBC of the hybrid Ag/PAA NPs. The antibacterial activities were also performed by cup diffusion method. Firstly, 100 μL bacteria suspensions were put uniformly on the surface of the agar Petri dish and solidified. Secondly, the sterile Oxford cups with a diameter of 6 mm were placed on the Petri dish and marked. Thirdly, the sample solutions with different concentrations were added into the Oxford cups. At the same time, the normal saline as control sample was dropped into the central cups. Finally, the agar Petri dishes were placed in the constant temperature incubator at 37 °C for 22 h. The antibacterial effect was evaluated by the diameter of visible transparent inhibitory zone. The antibacterial activity was also studied by the bacterial growth kinetics in broth media [21]. 2.5 mL of sample solutions with different concentrations (2, 40, 100 μg/mL) was mixed with 2.5 mL of sterilized broth media in 10 mL culture tubes. Then, 50 μL of bacterial suspensions was added into these tubes, and they were incubated at 37 °C for 48 h. These tubes were withdrawn at set time intervals to measure the optical density (OD) at 600 nm by using the UV–vis absorption spectrophotometer. As the control group, 50 μL of bacterial suspension was injected into 5 mL broth medium and performed to the same procedure as the
above. Finally, the OD values were plotted against the time to obtain the growth curve. In addition, the change of morphology of bacteria treated with Ag/PAA NPs was evaluated by SEM and TEM [22]. 2 mL of broth medium and 40 μL of bacterial suspensions were mixed and cultured at 37 °C for 6 h. Then, 100 μg/mL Ag/PAA NPs was added and allowed to culture at the same condition for another 6 h. At last, the bacteria were collected by centrifugation. To fabricate bacterial SEM and TEM samples, the bacteria were fixed with a diluted glutaraldehyde solution (2.5%) at −4 °C for 30 min and centrifuged at 6000 r/min for 5 min. After dehydration with series of alcohol solution, the collected bacteria were observed by SEM and TEM. 3. Results and discussion 3.1. XRD patterns of Ag/PAA NPs Fig. 1 shows that XRD patterns of the as-synthesized Ag/PAA NPs with different molar ratio of PAA/Ag. It is clear to see that the three samples present multiple diffraction peaks, and the peaks at 2θ of 38.1°, 44.3°, 64.4°, 77.4° and 81.5° are assigned to diffractions from the (111), (200), (220), (311) and (222) lattice planes of face-centered cubic (fcc) of silver (JCPDS No.04-0783), respectively. There are no diffraction peaks of Ag2O, which suggests that the existence of modification layer of PAA can prevent the oxidation of Ag NPs. 3.2. Morphologies of Ag/PAA NPs Fig. 2 shows the TEM images of the as-synthesized Ag/PAA NPs with different molar ratio of PAA/Ag, the insets are histograms of the particles size distribution. The histograms were measured and calculated from TEM images through size statistics software of scion image [23]. It can be seen from Fig. 2(b) when the molar ratio of PAA/Ag is 1:2, the sample exhibits uniform sphere shape, and have an average size of 8.5 nm with a size distribution of ±1.8 nm. While the molar ratio of PAA/Ag is 1:4, the obtained sample (Fig. 2(a)) is quasi sphere in shape. The average particles size and distribution is 8.8 and ±2.2 nm, respectively. When Table 1 MIC and MBC numerical values of Ag, Ag/PAA NPs and PAA. MIC (μg/mL)
Fig. 4. FTIR spectra of Ag/PAA NPs with different molar ratio of PAA/Ag (a) 1:4, (b) 1:2, (c) 1:1.
Ag PAA/Ag PAA
MBC (μg/mL)
E. coli
P. aeruginosa
S. aureus
E. coli
P. aeruginosa
S. aureus
125 3.9 ——
250 7.8 1000
125 3.9 1000
250 15.6 ——
500 62.5 ——
250 15.6 ——
——: indicating no antibacterial properties.
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Fig. 6. Inhibition zone photographs of Ag/PAA NPs against bacteria E. coli (a, b), P. aeruginosa (c, d) and S. aureus (e, f). (1–8) Samples correspond concentrations with 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL and the control group in the middle is 9% normal saline (NS).
the molar ratio of PAA/Ag increased to 1:1, obvious aggregated particles emerged, the mean particles size of 10.0 nm was estimated, and the particles size distribution of ± 2.2 nm was calculated. From TEM
observation, it can be concluded that the optimum molar ratio of PAA/Ag is 1:2, so this sample is chosen for thermal analysis and antibacterial test. 3.3. Optical properties of Ag/PAA NPs Fig. 3 shows UV–vis absorption spectra of the Ag/PAA NPs with different molar ratio of PAA/Ag. It can be seen that the appearance of peaks around 424 nm of three curves clearly indicates the formation of Ag NPs, which was attributed to the excitation of surface plasmon resonance (SPR) [24,25] or inter-band transition of silver NPs. The obtuse absorption peak in Fig. 3(a) is possibly assigned to little PAA coated on the surface and the wide particles size distribution of Ag/PAA NPs. It also can be found from Fig. 3(b) compared with Fig. 3(a) that a blue shift of the in-plane dipole resonance from 426 to 424 nm occurs, which suggests that the particles obtained here are truncated. 3.4. FTIR spectra of Ag/PAA NPs
Fig. 7. Growth curves of E. coli (A) and S. aureus (B) with different concentration Ag/PAA NPs treated (b, c, d) and bacterial without Ag/PAA as control test (a).
To gain further insight into the interaction of PAA with the Ag NPs surface, FTIR spectroscopy is used to analyze the organic functional groups. Fig. 4 shows FTIR spectra of Ag/PAA NPs with different molar ratio of PAA/Ag. As is shown in Fig. 4(b), a wide band at 3426 cm−1 is attributed to the stretching vibration of hydroxyl group in the adsorbed water. The band at 2927 cm−1 is assigned to the stretching vibration of –CH2 group from the long aliphatic chains. The band at 1630 cm−1 is assigned to stretching vibration of the free carbonyl groups. The bands at 1390 cm− 1 and 1260 cm− 1 can be assigned to the symmetric and anti-symmetric vibration modes of –COO− groups, indicating the adsorption of PAA on the nanoparticles through the bidentate bonds [26–28]. The wide band at 814 cm − 1 is the skeleton vibration of –C–C–. Compared with the relative intensity of each absorption bands at 1390, 1260 and 814 cm−1 in Fig. 4(a–c), it can be seen that the relative intensity gradually increased with the augment of molar ratio of PAA/Ag. The band at 679 cm−1 is assigned to stretching vibration of O-Ag [29,30], indicating that a coordination reaction between –COO and Ag atoms may occur.
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Fig. 8. SEM images of the normal and treated bacteria with 100 μg/mL of Ag/PAA NPs solution for E. coli (a, b), P. aeruginosa (c, d), and S. aureus (e, f).
3.5. Thermal analysis of Ag/PAA NPs Fig. 5 shows TG curves of the as-prepared PAA/Ag NPs with the molar ratio of PAA/Ag at 1:2 (a) and pure Ag particles without surface modification (b). It can be seen from Fig. 5(a) that Ag/PAA NPs exhibit obviously two weight loss processes. The first weight loss process (about 3%) below 100 °C may be attributed to the release of adsorbed water. The second mass loss process (about 15%) in the range of 160 °C to 560 °C is attributed to the decomposition of PAA binding on the nanoparticles. By comparison, for pure Ag NPs, there is almost no mass loss in the whole temperature raising range from ambient temperature to 800 °C. Accordingly, it can be concluded that PAA has been coated on the surfaces of Ag NPs through chemical reaction, which is consistent with the FTIR analysis. 3.6. Antimicrobial activities of Ag/PAA NP Table 1 shows the MIC and MBC values of Ag, Ag/PAA NPs, PAA against E. coli, S. aureus and P. aeruginosa measured by broth dilution method. The average particle size of Ag/PAA NPs is 8.5 nm (the sample with PAA/Ag molar ratio of 1:2), yet that of Ag NPs without PAA is about
Fig. 9. TEM images of the normal bacteria (a) and treated E. coli with 100 μg/mL of Ag/PAA NPs (b), the insets are partial enlarged images.
400 nm and the nanoparticles have severe aggregation. From Table 1, it can be seen that the MIC and MBC value of Ag against E. coli, S. aureus and P. aeruginosa is more than 125 μg/mL. PAA almost exhibits no antibacterial property. However, the MIC and MBC values of Ag/PAA NPs against E. coli, S. aureus and P. aeruginosa are lower than 7.8 and 62.5 μg/mL, respectively, which are obviously better than those of Ag and PAA. These results show that the Ag/PAA NPs have excellent antibacterial properties. From Table 1, it also can be seen that Ag/PAA NPs exhibit equal antibacterial against Gram-negative bacteria of E. coli and Gram-positive bacteria of S. aureus, which is some different from the reference [31]. This may be attributed to the Ag NPs size and stable agents are different in the two work. Fig. 6 shows inhibition zone photographs of Ag/PAA NPs against E. coli (a, b), P. aeruginosa (c, d), S. aureus (e, f), respectively. Samples (1–8) corresponds to the concentrations with 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL and the control group in the middle was 9% normal saline (NS) [19,32]. Measurement results show that the diameter of inhibition zone of Ag/PAA NPs against E. coli, P. aeruginosa and S. aureus are 10.0, 16.0 and 20.0 mm at the sample concentration 125 μg/mL, respectively. There is no obvious inhibition zone for the control test of NS. Besides the MIC measurement, the inhibition zone test results further indicate intuitively that the as-synthesized Ag/PAA NPs exhibit bacterial inhibition effects. The antibacterial properties were also investigated by testing the bacterial growth curves in liquid broth media. The time-dependent changes of bacterial growth were monitored by measuring OD600. Fig. 7 shows the growth curves of typical Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus) with different concentration Ag/PAA NPS for 48 h, the control test is bacteria in broth medium without Ag/PAA NPs. It is clearly to see that the sample at all tested concentrations has strong suppression of proliferation of tested strains. For E. coli, when the concentration was over 20 μg/mL, the sample could inhibit completely the growth of E. coli during the whole 48 h (Fig. 7(a)). However, when the concentration was below MIC (3.9 μg/mL), it was not enough to inhibit the growth of E. coli within 48 h, and there was a growth delay of E. coli. For S. aureus, the solution of 20 μg/mL and 60 μg/mL could completely inhibit the growth of bacteria. Similarly, the growth curves of S. aureus with the concentration 2 μg/mL of
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Ag/PAA NPs also showed a lag phase compared to the control test of S. aureus (Fig. 7(b)). The influence of Ag/PAA solution to bacteria was further evaluated by SEM through observing the change of morphology of the original and treated bacteria. Fig. 8 shows SEM images of the normal and treated bacteria with 100 μg/mL of Ag/PAA NPs solution for E. coli (a, b), P. aeruginosa (c, d), and S. aureus (e, f). It can be seen from Fig. 8(a) that original E. coli exhibit uniform short rod morphologies with smooth surfaces while as shown in Fig. 8(b), the treated E. coli cells exhibit significantly changes in reduced length and rough surfaces, which indicates the occurrence of interaction of Ag/PAA NPs and the distinct damage of bacterial cells. The similar phenomenon can be observed by comparison of Fig. 8(d) and (e) of untreated and treated bacteria of P. aeruginosa. The normal S. aureus cells observed from Fig. 8(e) is spherical in shape with an average diameter of 1 μm and has smooth cell surfaces, whereas the membrane deformation and rough surfaces formation can be seen along with the appearance of cell debris in the case of treated cells as shown in Fig. 8(f). In order to demonstrate further the integration of Ag/PAA NPs and cell wall, E. coli was chosen as a typical bacterium for TEM observation. Fig. 9 shows TEM images of the normal E. coli (a) and treated E. coli with 100 μg/mL of Ag/PAA NPs (b), the insets are partial enlarged images. It is obvious to see from Fig. 9 that after treated with Ag/PAA NPs, the rodlike cell of E. coli broke into fragments, and Ag/PAA NPs were attached on the surfaces of the bacterial cell. Although the exact action mechanism of Ag NPs against bacteria is not very well known, the possible antibacterial action model can be proposed based the above‐mentioned results. Ag/PAA NPs can attach to the surface of the cell membrane and disturb its functions, and then it penetrates the bacteria to cause further damage, such as leakage of cytoplasm. In addition, Ag/PAA NPs may release Ag+, which make an additional contribution to the bactericidal effect [33,34]. 4. Conclusions Ag NPs modified by PAA is successfully synthesized by chemical reduction method. The particles’ morphologies and sizes were depended strongly on the molar ratio of surfactant agent to Ag. The Ag/PAA NPs have excellent water solubility, stability and biological compatibility. Ag/PAA NPs exhibit effective bacteriostasis and bactericidal activity against E. coli, S. aureus and P. aeruginosa. The possible antibacterial mechanism against bacteria is proposed. Acknowledgements The authors are grateful for the financial support provided by National Natural Science Foundation of China (50701016, 21271062), China
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