Materials Science and Engineering C 49 (2015) 316–322
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Antibacterial activity of silver nanoparticles synthesized from serine N. Jayaprakash a,b, J. Judith Vijaya a,⁎, L. John Kennedy c, K. Priadharsini d, P. Palani d a
Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034, India SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203, India c Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048, India d Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025, India b
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Article history: Received 16 April 2014 Received in revised form 24 December 2014 Accepted 4 January 2015 Available online 7 January 2015 Keywords: Silver nanoparticles Serine Photoluminescence Electron microscopy Microwave irradiation
a b s t r a c t Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Metallic nanoparticles are used in different fields, such as catalysis, media recording, biosensing, optics, environmental remediation, biomedical, pharmaceutical, cosmetic, energy, electronic devices, imaging, drug delivery, and medicine [1–4]. Specific size, shape and surface morphology of the metallic nanoparticles play a vital role in controlling the properties of the nanoscopic materials [5,6]. Different synthetic methods are reported in the literature for the preparation of the metallic nanoparticles [7,8]. Though conventional (chemical and physical) methods are available to synthesize the metallic nanoparticles, they are expensive and required more time. Thus, there is an increasing need to develop the high-yield, low cost, non-toxic and eco-friendly procedures for the synthesis of metallic nanoparticles. Among the various noble metal nanoparticles, Ag NPs have shown essential applications in various fields like catalysis, bio-sensing, imaging, drug delivery, nano-device fabrication and medicine [9–11]. Due to the strong anti-microbial activity, Ag NPs are also used in clothing, food industry, sunscreens and cosmetics [12–14]. Hence, Ag NPs have been projected as the future generation antimicrobial agents [15]. Oxidation of metallic silver is thermodynamically unfavorable, due to its higher positive reduction potential and thus, resulting in quite stable in aqueous and alcoholic suspensions without the aid of capping agents. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Judith Vijaya).
http://dx.doi.org/10.1016/j.msec.2015.01.012 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Chemical reduction methods are commonly used to synthesize the Ag NPs. Typical reducing agents, such as polyols [16,17], NaBH4 [18–20], N2H4 [21], sodium citrate [22], polymeric thiols [23], Triton X 100 [2], and amino acids [24–27] are used to synthesize the Ag NPs. Polyvinylpyrrolidone (PVP) and polyvinyl alcohol are eco-friendly polymers, since they are water soluble, and have extremely low cytotoxicity. These polymeric substances are frequently used as stabilizers, due to their optical clarity, which enables the investigation of the formation of nanoparticles [28,29]. The antibacterial activities of PVP modified Ag NPs are significant because the PVP is most effective in stabilizing the particles against aggregation [30]. Most of the chemical reduction reactions are carried out at high temperatures for a higher reaction rate, and it can be done by conventional heating [16,17], laser irradiation [31,32], ultrasonic [33], fixed frequency microwave radiation [34, 35], UV irradiation [36], and gamma-ray irradiation method [37]. Several methods for the synthesis of Ag NPs have been reported in the literature [38,39]. However, microwave irradiation method is known to have a faster heating rate than the conventional heating. The use of a fixed frequency microwave to synthesize platinum and silver nanoparticles is also reported [34,35,40]. Microwave irradiation method is more eco-friendly and requires less energy than the other conventional methods. This method offers rapid and uniform heating of solvents, reagents, and intermediates. Further, it provides uniform nucleation and growth conditions for the nanomaterials [27]. In the present work, we have described the synthesis of Ag NPs by a microwave irradiation method using serine as a reducing and PVP as a
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capping agent in aqueous medium. Thus, a very simple, low cost and green way to synthesize Ag NPs and the antibacterial activity studies are reported herein.
2. Experimental 2.1. Chemicals Silver nitrate (AgNO3), serine and PVP (Avg. mol. wt 40,000) were purchased from Qualigens Fine Chemicals, Mumbai, India, and were used without further purification for the synthesis of Ag NPs. De-ionized water was used for the synthesis and characterization of Ag NPs.
2.2. Characterization techniques The UV–Vis spectra were recorded by a Shimadzu 1800 spectrophotometer. Morphology was determined by using Jeol 200 CX highresolution transmission electron microscopy (HR-TEM) and FE I Quata FEG 200 high-resolution scanning electron microscopy (HR-SEM). The sample for HR-TEM analysis was prepared by placing a drop of the solution on a carbon coated copper grid and dried in air. Elemental composition was obtained from energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. Emission spectrum of the Ag NPs was recorded by using a Varian Cary Eclipse fluorospectrometer. Fourier transform infrared (FT-IR) spectrum was obtained from a Fourier transform infrared spectrometer (BRUKER, Alpha T model). For FT-IR spectrum, Ag NPs were separated by centrifugation at 10,000 rpm for 15 min. The sample was dried, ground with KBr and made as a pellet which was analyzed by an FT-IR spectrometer. Electrochemical measurements were made with a CHI 600A electrochemical workstation controlled by a personnel computer. Three electrode systems were employed in this study. A platinum wire, glassy carbon electrode (GCE) and a saturated calomel electrode (SCE) were used as counter electrode, working electrode and reference electrode respectively. 0.1 M KNO3 was used as an electrolytic solution. 2.3. Synthesis of silver nanoparticles A microwave oven (model: MS-2049 UW) was used in the typical synthesis of Ag NPs. 100 mL of 5 mM silver nitrate solution was prepared in 250 mL conical flask using the de-ionized water. 0.0525 g of serine and 0.75 g of PVP were added into the above-mentioned silver nitrate solution, and then 4 drops of 1 M NaOH were added by altering the pH for the favorable reduction. The above mixed solution was kept in the microwave oven (input power 1200 W, 50 Hz) for 90 s. The solution turned into yellowish brown color, which confirmed the formation of Ag NPs. Fig. 1 shows the digital photo of the synthesized Ag NP colloidal solutions at different time intervals of 15, 30, 45, 60, 75, and 90 s.
Fig. 1. Digital photo of the synthesized Ag NP colloidal solutions at different time intervals of 15, 30, 45, 60, 75, and 90 s.
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2.4. Antibacterial activity The Ag NPs in sterilized distilled water were tested for their antibacterial activity by the agar diffusion method or modified Kirby–Bauer method. Nine bacterial strains, Bacillus cereus, Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, and Enterococcus as Gram-positive bacteria and Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Klebsiella pneumonia as Gram-negative bacteria were used for the antibacterial activity analysis. All human pathogens used in the antibacterial study were obtained from the Department of Medical Microbiology, Taramani Campus, University of Madras, India. These bacteria were grown on liquid nutrient agar media for 24 h prior to the experiment, and were seeded in agar plates by the pour plate technique. Different plates were prepared for every bacterial strain. In each petri plate, four cavities were made using a cork borer at an equal distance. In each cavity, 50 μL of Ag NPs, serine, AgNO3 and PVP solutions was filled. All the plates were incubated at 35 °C for 24 h. All the experiments were repeated thrice to ensure the reproducibility. 3. Results and discussion 3.1. UV–Visible spectroscopic studies of Ag NPs UV–Visible absorption spectra provide the information about the formation, particle size and surface properties of Ag NPs. Fig. 2 shows the UV–Visible absorption spectra of the solutions containing synthesized Ag NPs, serine, PVP, and AgNO3. The formation of Ag NPs was confirmed by the presence of an intense peak at 443 nm. The obtained absorption spectrum of Ag NP colloidal solution is similar with that of our earlier report for the Ag nanospheres [2]. According to Mie's theory [41], spherical Ag NPs will give a single symmetric absorption peak, but anisotropic Ag NPs will exhibit two or more bands. Moreover, the UV–Visible spectrum of Ag NPs was symmetrical in nature, which suggested that the synthesized Ag NPs had sphere-like morphology. Fig. 3 shows the UV–Visible absorption spectra of the solutions containing synthesized Ag NPs at different time intervals of 30, 45, 60, 75, 90, and 120 s. The UV–Visible absorption spectra confirmed the formation of Ag NPs at 90 s. There was no much difference in the spectra beyond 90 s. Fig. 4 displays the absorbance versus time plot at 5 mM of Ag NPs. It showed that the curve was sigmoid in nature, which suggested that the reaction had complicated kinetic features [25]. Fig. 5 shows the plot of ln[a / (1 − a)] against time, where a = At / A∞, and At and A∞ are the absorbance at time t and infinite time respectively. Fig. 5 indicates the involvement of the autocatalytic reaction paths in the formation of Ag NPs. The occurrence of autocatalytic reaction might be
Fig. 2. UV–Visible absorption spectra of the solutions containing synthesized Ag NPs, serine, PVP, and AgNO3.
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Fig. 3. UV–Visible absorption spectra of the solutions containing synthesized Ag NPs at different time intervals of 30, 45, 60, 75, 90, and 120 s.
Fig. 5. Plot of ln[a / (1 − a)] against microwave irradiation time.
due to the formation of Ag0 nucleation center, which acts as a catalyst for the reduction of other Ag+ ions present in the solution [25].
2944 cm− 1, 1690 cm− 1, 1536 cm− 1, 1245 cm− 1, 1054 cm−1 and 605 cm−1 corresponding to OH stretching, CH2 asymmetric stretching, asymmetric deformation,\COO− asymmetric stretching, \NH+ 3 C\OH rocking, C\N asymmetric stretching, and COO− rocking respectively. FT-IR spectrum of Ag NPs was similar to that of serine with a slight shift in the band positions and also increased in the band intensities of the functional groups, which clearly indicated the presence of the reaction. It showed the bands at 3321 cm−1, 2925 cm−1, 2121 cm−1, 1658 cm−1, 1122 cm−1, 752 cm−1, and 604 cm−1, which were assigned to OH stretching, C\H stretching, N\H stretching, C_O stretching, C\OH rocking, C\H out-of-plane deformation, and COO− rocking respectively. The increase in the band intensity of C_O group suggested that the \OH group was oxidized to C_O, during the reaction, and could be inferred as responsible for the formation of Ag NPs [44–48].
3.2. Fluorescence spectroscopic study of Ag NPs Photoluminescence analysis was carried out to understand the fluorescence property of the synthesized Ag NPs. The synthesized Ag NPs were excited at 443 nm, and the emission peak was obtained at 484 nm (Fig. 6). Gao et al. [42] reported the emission band at 486 nm on excitation at 408 nm for Ag NPs stabilized by [poly(styrene)]dibenzo-18-crown-6-[poly(styrene)]. Angshuman et al. [43] also reported the emission band at 491 nm on excitation at 416 nm for Ag NPs stabilized by PVP. We have recently reported the emission band at 482 nm on excitation at 414 nm for silver nanospheres stabilized by Triton X 100 [2]. The origin of the fluorescence could be attributed to the promotion of d-band electrons of the silver nanoparticles on absorption of the incident radiation to higher electronic states in the sp-band.
3.4. Microscopic analysis
The FT-IR spectrum of Ag NPs was analyzed in order to identify the functional groups of serine involved in the reduction of AgNO3. Fig. 7(a–b) shows the FT-IR spectrum of pure serine and Ag NPs. FT-IR spectrum of pure serine showed the prominent bands at 3323 cm−1,
HR-SEM images had provided further details about the morphology and the size of the Ag NPs. The HR-SEM images of Ag NPs with different magnifications are shown in Fig. 8. These images confirmed the formation of highly dispersed sphere-like particles. The higher magnified HR-SEM image (Fig. 8a–b) suggested that the silver microspheres were composed of nanoparticles. Also, the surface of the microspheres was highly porous in nature (Fig. 8c–d). The formation of Ag NPs was
Fig. 4. Plot of absorbance versus microwave irradiation time.
Fig. 6. Emission spectrum of Ag NPs.
3.3. Fourier transform infrared spectroscopy
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Fig. 9. HR-TEM image of Ag NPs. Fig. 7. FT-IR spectrum of pure serine (a) and Ag NPs (b).
further confirmed by HR-TEM image (Fig. 9). HR-TEM image revealed that the size of the Ag NPs might be around 65 nm. The possible formation mechanism of silver microspheres was explained as follows. The Ag NPs were synthesized by the reduction of Ag+ ions in the presence of microwave radiation using serine as a reducing agent and PVP as a capping agent. The size of silver nanoparticles can be controlled not only by the mass ratio of the reducing agent to Ag+ ions, but also by pH alteration. The stability of silver nanoparticles and reactive species of the reductant strongly depends on the pH of the working solutions. Zwitterionic species is a major reactive species of serine. As serine contains both acidic and basic functional groups, at pH below the isoelectric point (pI, 5.68), serine carries a net positive charge, i.e. the \NH2 group has partly changed into \NH+ 3 . Complete protonation of the carboxyl and amino groups lowers the reduction potential to such an extent that the oxidation site may effectively remain at the \OH group. Above pI of 5.68, serine carries a net negative charge, i.e. the \COOH group has changed into \COO−. Hence, the electrondonating ability of serine would be greatly changed. Serine reduces
Ag+ to Ag NPs. This reaction is more favorable by adjusting the pH using 1 M NaOH. Thereafter, the pH of the working solution is measured and found to be constant (pH = 6.91 ± 0.2) under different experimental conditions. The reaction might be the oxidation of \OH group of serine into aldehyde group [2,25,49] by reducing Ag+ (Scheme 1, Eq. (1)). Or else, the reaction might be the electron transfer from the \OH group of serine to Ag+ leading to the formation of Ag0 nuclei, which undergo coalescent [50] processes to form particles and serine radical. Due to the low colloidal stability, Ag0 nanoparticles undergo agglomeration to form Ag0 microspheres. Serine radicals may undergo dimerization [25] (Scheme 1, Eqs. (2) to (4)). The graphical representation of the formation of Ag microspheres is shown in Scheme 2. 3.5. Energy dispersive X-ray analysis and SAED studies The EDX spectrum was recorded to determine the elements present in the reaction product. The EDX spectrum (Fig. 10) showed different peaks for the presence of Ag, O and C. The Ag, O and C peaks came
Fig. 8. HR-SEM images of Ag NPs at magnifications of (a) 5 μm, (b) 2 μm, (c) 1 μm, and (d) 500 nm.
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Scheme 1. Possible formation mechanism of silver nanoparticles using serine as a reducing agent.
Scheme 2. Graphical representation for the formation mechanism of silver microspheres.
from Ag NPs, carbonyl group of serine and PVP respectively. Metallic silver nanocrystals generally have shown an optical absorption peak approximately at 3 keV, due to the surface plasmon resonance [51]. The SAED pattern (Fig. 11) of Ag NPs prepared by microwave irradiation method showed concentric rings, which resulted from the random orientation of the crystal planes. This suggested that the sample was nano-crystalline in nature.
Fig. 12 shows the cyclic voltammogram (CV) of serine and PVP capped Ag NPs recorded at 0.05 Vs−1. Fig. 12a shows the redox peaks in the potential range of −0.6 to +1.2 V, due to the redox active components of serine. The presence of oxidation peak suggested that the components of serine might be gaining electrons from the electrode.
Fig. 10. Energy dispersive X-ray analysis of Ag NPs.
Fig. 11. SAED pattern of Ag NPs.
3.6. Cyclic voltammetry
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Fig. 12. Cyclic voltammogram (CV) of (a) pure serine and (b) PVP capped Ag NPs.
The reduction peak was due to its oxidation in the presence of an electric field. The CV of PVP capped Ag NPs (Fig. 12b) showed a well defined redox peak and was similar to that of serine. Further, additional oxidation and reduction peaks corresponding to the redox behavior of Ag0 were also observed [52,53]. The observed electrochemical activity shows that the prepared Ag NPs will have great potential in the field of electrochemical sensors [54]. 3.7. Antimicrobial activity of Ag NPs against microorganisms The synthesized Ag NP colloidal solution showed antibacterial activity against Gram-positive bacteria such as B. cereus, S. aureus, M. luteus,
Fig. 14. Bar diagram of inhibition zones of antibacterial activity of Ag NPs and AgNO3.
B. subtilis and Enterococcus and also against Gram-negative bacteria such as P. aeruginosa, S. typhi, E. coli, and K. pneumonia by showing the inhibition zones around the holes with bacteria growth on petri plates by agar diffusion method. Fig. 13 shows the result of inhibition zones of antibacterial activity. The diameters of the inhibiting zones of Ag NPs against B. cereus, S. aureus, M. luteus, B. subtilis, and Enterococcus were 19, 16, 36, 19, and 19 mm respectively for Gram-positive bacteria and P. aeruginosa, S. typhi, E. coli, and K. pneumonia were 21, 21, 21, and 12 mm respectively
Fig. 13. Results of inhibition zones of antibacterial activity (1. Ag NPs, 2. AgNO3, 3. Serine, and 4. PVP). (a) Bacillus cereus, (b) Staphylococcus aureus, (c) Pseudomonas aeruginosa, (d) Salmonella typhi, (e) Micrococcus luteus, (f) Escherichia coli, (g) Klebsiella pneumoniae, (h) Bacillus subtilis, and (i) Enterococcus.
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for Gram-negative bacteria. The diameters of the inhibiting zones of AgNO3 against B. cereus, S. aureus, M. luteus, B. subtilis and Enterococcus were 18, 14, 35, 18, 18 mm respectively for Gram-positive bacteria and P. aeruginosa, S. typhi, E. coli, and K. pneumonia were 20, 21, 20, and 13 mm respectively for Gram-negative bacteria. At the same time, the cavities containing serine and PVP respectively did not show any appreciable inhibition zones. The above given results were the mean value of the three separate experiments conducted. The inhibition zones of Ag NPs shown in the present study were found to be relatively similar, when compared with AgNO3 against Gram-positive and Gramnegative bacteria. There was a recent report [55] regarding the Ag NPs against some bacteria, which had been used by us. But, they showed lesser inhibition zone than our present synthesized Ag NPs at the same 50 μL concentration. The inhibition zone of Ag NPs against M. luteus was higher than the other bacteria. Fig. 14 shows the bar diagram of inhibition zones of Ag NPs and AgNO3. The bactericidal activity of Ag NPs is certainly due to the silver cations released from Ag NPs that act as reservoirs for the Ag+ bactericidal agent [55]. Ag+ ions are supposed to bind to sulfhydryl groups, which leads to the protein denaturation by the reduction of disulfide bonds. Also, Ag+ can form complex with the electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids. Hence, Ag NPs might have been attached to the surface of the cell membrane to disturb its function, penetrate into bacteria, and release Ag [3]. Normally several silver containing salts showed a good antimicrobial activity. But, the higher concentration of silver is harmful to both the consumers and the microbes. Hence, the smaller concentration like nano-range is much more applicable for this purpose. The present microwave-assisted synthesis of Ag NPs will be more helpful for pharmaceutical and medical applications.
[6] [7] [8] [9] [10] [11]
4. Conclusion
[35]
Ag NPs were successfully synthesized by using serine as a reducing agent and PVP as a capping agent. The hydroxyl group of serine was involved in the reduction of Ag+ ions into Ag NPs. UV–Visible, PL, FT-IR, HR-SEM, HR-TEM, EDX, SAED and CV were used to characterize the prepared Ag NPs. The synthesized Ag NPs had found to be extremely stable for more than 6 months. This present method has more advantages like cost effective, less reaction time, formation of highly stable nanoparticles, straightforward and scalable. Also, the synthesized Ag NPs could find more valuable applications in the field of sensor, medicine and environmental remedies. Acknowledgments We thank Nanotechnology Research Centre (for HR-SEM with EDX) and Interdisciplinary School of Indian System of Medicine (for FT-IR) SRM University, Chennai, India. We also heartily thank Dr. L. Devaraj Stephen for his help to record HR-TEM from IISc, Bangalore, India.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Antibacterial activity of silver nanoparticles synthesized from serine N. Jayaprakash a,b, J. Judith Vijaya a,⁎, L. John Kennedy c, K. Priadharsini d, P. Palani d a
Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034, India SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203, India c Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048, India d Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025, India b
a r t i c l e
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Article history: Received 16 April 2014 Received in revised form 24 December 2014 Accepted 4 January 2015 Available online 7 January 2015 Keywords: Silver nanoparticles Serine Photoluminescence Electron microscopy Microwave irradiation
a b s t r a c t Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Metallic nanoparticles are used in different fields, such as catalysis, media recording, biosensing, optics, environmental remediation, biomedical, pharmaceutical, cosmetic, energy, electronic devices, imaging, drug delivery, and medicine [1–4]. Specific size, shape and surface morphology of the metallic nanoparticles play a vital role in controlling the properties of the nanoscopic materials [5,6]. Different synthetic methods are reported in the literature for the preparation of the metallic nanoparticles [7,8]. Though conventional (chemical and physical) methods are available to synthesize the metallic nanoparticles, they are expensive and required more time. Thus, there is an increasing need to develop the high-yield, low cost, non-toxic and eco-friendly procedures for the synthesis of metallic nanoparticles. Among the various noble metal nanoparticles, Ag NPs have shown essential applications in various fields like catalysis, bio-sensing, imaging, drug delivery, nano-device fabrication and medicine [9–11]. Due to the strong anti-microbial activity, Ag NPs are also used in clothing, food industry, sunscreens and cosmetics [12–14]. Hence, Ag NPs have been projected as the future generation antimicrobial agents [15]. Oxidation of metallic silver is thermodynamically unfavorable, due to its higher positive reduction potential and thus, resulting in quite stable in aqueous and alcoholic suspensions without the aid of capping agents. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Judith Vijaya).
http://dx.doi.org/10.1016/j.msec.2015.01.012 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Chemical reduction methods are commonly used to synthesize the Ag NPs. Typical reducing agents, such as polyols [16,17], NaBH4 [18–20], N2H4 [21], sodium citrate [22], polymeric thiols [23], Triton X 100 [2], and amino acids [24–27] are used to synthesize the Ag NPs. Polyvinylpyrrolidone (PVP) and polyvinyl alcohol are eco-friendly polymers, since they are water soluble, and have extremely low cytotoxicity. These polymeric substances are frequently used as stabilizers, due to their optical clarity, which enables the investigation of the formation of nanoparticles [28,29]. The antibacterial activities of PVP modified Ag NPs are significant because the PVP is most effective in stabilizing the particles against aggregation [30]. Most of the chemical reduction reactions are carried out at high temperatures for a higher reaction rate, and it can be done by conventional heating [16,17], laser irradiation [31,32], ultrasonic [33], fixed frequency microwave radiation [34, 35], UV irradiation [36], and gamma-ray irradiation method [37]. Several methods for the synthesis of Ag NPs have been reported in the literature [38,39]. However, microwave irradiation method is known to have a faster heating rate than the conventional heating. The use of a fixed frequency microwave to synthesize platinum and silver nanoparticles is also reported [34,35,40]. Microwave irradiation method is more eco-friendly and requires less energy than the other conventional methods. This method offers rapid and uniform heating of solvents, reagents, and intermediates. Further, it provides uniform nucleation and growth conditions for the nanomaterials [27]. In the present work, we have described the synthesis of Ag NPs by a microwave irradiation method using serine as a reducing and PVP as a
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capping agent in aqueous medium. Thus, a very simple, low cost and green way to synthesize Ag NPs and the antibacterial activity studies are reported herein.
2. Experimental 2.1. Chemicals Silver nitrate (AgNO3), serine and PVP (Avg. mol. wt 40,000) were purchased from Qualigens Fine Chemicals, Mumbai, India, and were used without further purification for the synthesis of Ag NPs. De-ionized water was used for the synthesis and characterization of Ag NPs.
2.2. Characterization techniques The UV–Vis spectra were recorded by a Shimadzu 1800 spectrophotometer. Morphology was determined by using Jeol 200 CX highresolution transmission electron microscopy (HR-TEM) and FE I Quata FEG 200 high-resolution scanning electron microscopy (HR-SEM). The sample for HR-TEM analysis was prepared by placing a drop of the solution on a carbon coated copper grid and dried in air. Elemental composition was obtained from energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. Emission spectrum of the Ag NPs was recorded by using a Varian Cary Eclipse fluorospectrometer. Fourier transform infrared (FT-IR) spectrum was obtained from a Fourier transform infrared spectrometer (BRUKER, Alpha T model). For FT-IR spectrum, Ag NPs were separated by centrifugation at 10,000 rpm for 15 min. The sample was dried, ground with KBr and made as a pellet which was analyzed by an FT-IR spectrometer. Electrochemical measurements were made with a CHI 600A electrochemical workstation controlled by a personnel computer. Three electrode systems were employed in this study. A platinum wire, glassy carbon electrode (GCE) and a saturated calomel electrode (SCE) were used as counter electrode, working electrode and reference electrode respectively. 0.1 M KNO3 was used as an electrolytic solution. 2.3. Synthesis of silver nanoparticles A microwave oven (model: MS-2049 UW) was used in the typical synthesis of Ag NPs. 100 mL of 5 mM silver nitrate solution was prepared in 250 mL conical flask using the de-ionized water. 0.0525 g of serine and 0.75 g of PVP were added into the above-mentioned silver nitrate solution, and then 4 drops of 1 M NaOH were added by altering the pH for the favorable reduction. The above mixed solution was kept in the microwave oven (input power 1200 W, 50 Hz) for 90 s. The solution turned into yellowish brown color, which confirmed the formation of Ag NPs. Fig. 1 shows the digital photo of the synthesized Ag NP colloidal solutions at different time intervals of 15, 30, 45, 60, 75, and 90 s.
Fig. 1. Digital photo of the synthesized Ag NP colloidal solutions at different time intervals of 15, 30, 45, 60, 75, and 90 s.
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2.4. Antibacterial activity The Ag NPs in sterilized distilled water were tested for their antibacterial activity by the agar diffusion method or modified Kirby–Bauer method. Nine bacterial strains, Bacillus cereus, Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, and Enterococcus as Gram-positive bacteria and Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Klebsiella pneumonia as Gram-negative bacteria were used for the antibacterial activity analysis. All human pathogens used in the antibacterial study were obtained from the Department of Medical Microbiology, Taramani Campus, University of Madras, India. These bacteria were grown on liquid nutrient agar media for 24 h prior to the experiment, and were seeded in agar plates by the pour plate technique. Different plates were prepared for every bacterial strain. In each petri plate, four cavities were made using a cork borer at an equal distance. In each cavity, 50 μL of Ag NPs, serine, AgNO3 and PVP solutions was filled. All the plates were incubated at 35 °C for 24 h. All the experiments were repeated thrice to ensure the reproducibility. 3. Results and discussion 3.1. UV–Visible spectroscopic studies of Ag NPs UV–Visible absorption spectra provide the information about the formation, particle size and surface properties of Ag NPs. Fig. 2 shows the UV–Visible absorption spectra of the solutions containing synthesized Ag NPs, serine, PVP, and AgNO3. The formation of Ag NPs was confirmed by the presence of an intense peak at 443 nm. The obtained absorption spectrum of Ag NP colloidal solution is similar with that of our earlier report for the Ag nanospheres [2]. According to Mie's theory [41], spherical Ag NPs will give a single symmetric absorption peak, but anisotropic Ag NPs will exhibit two or more bands. Moreover, the UV–Visible spectrum of Ag NPs was symmetrical in nature, which suggested that the synthesized Ag NPs had sphere-like morphology. Fig. 3 shows the UV–Visible absorption spectra of the solutions containing synthesized Ag NPs at different time intervals of 30, 45, 60, 75, 90, and 120 s. The UV–Visible absorption spectra confirmed the formation of Ag NPs at 90 s. There was no much difference in the spectra beyond 90 s. Fig. 4 displays the absorbance versus time plot at 5 mM of Ag NPs. It showed that the curve was sigmoid in nature, which suggested that the reaction had complicated kinetic features [25]. Fig. 5 shows the plot of ln[a / (1 − a)] against time, where a = At / A∞, and At and A∞ are the absorbance at time t and infinite time respectively. Fig. 5 indicates the involvement of the autocatalytic reaction paths in the formation of Ag NPs. The occurrence of autocatalytic reaction might be
Fig. 2. UV–Visible absorption spectra of the solutions containing synthesized Ag NPs, serine, PVP, and AgNO3.
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Fig. 3. UV–Visible absorption spectra of the solutions containing synthesized Ag NPs at different time intervals of 30, 45, 60, 75, 90, and 120 s.
Fig. 5. Plot of ln[a / (1 − a)] against microwave irradiation time.
due to the formation of Ag0 nucleation center, which acts as a catalyst for the reduction of other Ag+ ions present in the solution [25].
2944 cm− 1, 1690 cm− 1, 1536 cm− 1, 1245 cm− 1, 1054 cm−1 and 605 cm−1 corresponding to OH stretching, CH2 asymmetric stretching, asymmetric deformation,\COO− asymmetric stretching, \NH+ 3 C\OH rocking, C\N asymmetric stretching, and COO− rocking respectively. FT-IR spectrum of Ag NPs was similar to that of serine with a slight shift in the band positions and also increased in the band intensities of the functional groups, which clearly indicated the presence of the reaction. It showed the bands at 3321 cm−1, 2925 cm−1, 2121 cm−1, 1658 cm−1, 1122 cm−1, 752 cm−1, and 604 cm−1, which were assigned to OH stretching, C\H stretching, N\H stretching, C_O stretching, C\OH rocking, C\H out-of-plane deformation, and COO− rocking respectively. The increase in the band intensity of C_O group suggested that the \OH group was oxidized to C_O, during the reaction, and could be inferred as responsible for the formation of Ag NPs [44–48].
3.2. Fluorescence spectroscopic study of Ag NPs Photoluminescence analysis was carried out to understand the fluorescence property of the synthesized Ag NPs. The synthesized Ag NPs were excited at 443 nm, and the emission peak was obtained at 484 nm (Fig. 6). Gao et al. [42] reported the emission band at 486 nm on excitation at 408 nm for Ag NPs stabilized by [poly(styrene)]dibenzo-18-crown-6-[poly(styrene)]. Angshuman et al. [43] also reported the emission band at 491 nm on excitation at 416 nm for Ag NPs stabilized by PVP. We have recently reported the emission band at 482 nm on excitation at 414 nm for silver nanospheres stabilized by Triton X 100 [2]. The origin of the fluorescence could be attributed to the promotion of d-band electrons of the silver nanoparticles on absorption of the incident radiation to higher electronic states in the sp-band.
3.4. Microscopic analysis
The FT-IR spectrum of Ag NPs was analyzed in order to identify the functional groups of serine involved in the reduction of AgNO3. Fig. 7(a–b) shows the FT-IR spectrum of pure serine and Ag NPs. FT-IR spectrum of pure serine showed the prominent bands at 3323 cm−1,
HR-SEM images had provided further details about the morphology and the size of the Ag NPs. The HR-SEM images of Ag NPs with different magnifications are shown in Fig. 8. These images confirmed the formation of highly dispersed sphere-like particles. The higher magnified HR-SEM image (Fig. 8a–b) suggested that the silver microspheres were composed of nanoparticles. Also, the surface of the microspheres was highly porous in nature (Fig. 8c–d). The formation of Ag NPs was
Fig. 4. Plot of absorbance versus microwave irradiation time.
Fig. 6. Emission spectrum of Ag NPs.
3.3. Fourier transform infrared spectroscopy
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Fig. 9. HR-TEM image of Ag NPs. Fig. 7. FT-IR spectrum of pure serine (a) and Ag NPs (b).
further confirmed by HR-TEM image (Fig. 9). HR-TEM image revealed that the size of the Ag NPs might be around 65 nm. The possible formation mechanism of silver microspheres was explained as follows. The Ag NPs were synthesized by the reduction of Ag+ ions in the presence of microwave radiation using serine as a reducing agent and PVP as a capping agent. The size of silver nanoparticles can be controlled not only by the mass ratio of the reducing agent to Ag+ ions, but also by pH alteration. The stability of silver nanoparticles and reactive species of the reductant strongly depends on the pH of the working solutions. Zwitterionic species is a major reactive species of serine. As serine contains both acidic and basic functional groups, at pH below the isoelectric point (pI, 5.68), serine carries a net positive charge, i.e. the \NH2 group has partly changed into \NH+ 3 . Complete protonation of the carboxyl and amino groups lowers the reduction potential to such an extent that the oxidation site may effectively remain at the \OH group. Above pI of 5.68, serine carries a net negative charge, i.e. the \COOH group has changed into \COO−. Hence, the electrondonating ability of serine would be greatly changed. Serine reduces
Ag+ to Ag NPs. This reaction is more favorable by adjusting the pH using 1 M NaOH. Thereafter, the pH of the working solution is measured and found to be constant (pH = 6.91 ± 0.2) under different experimental conditions. The reaction might be the oxidation of \OH group of serine into aldehyde group [2,25,49] by reducing Ag+ (Scheme 1, Eq. (1)). Or else, the reaction might be the electron transfer from the \OH group of serine to Ag+ leading to the formation of Ag0 nuclei, which undergo coalescent [50] processes to form particles and serine radical. Due to the low colloidal stability, Ag0 nanoparticles undergo agglomeration to form Ag0 microspheres. Serine radicals may undergo dimerization [25] (Scheme 1, Eqs. (2) to (4)). The graphical representation of the formation of Ag microspheres is shown in Scheme 2. 3.5. Energy dispersive X-ray analysis and SAED studies The EDX spectrum was recorded to determine the elements present in the reaction product. The EDX spectrum (Fig. 10) showed different peaks for the presence of Ag, O and C. The Ag, O and C peaks came
Fig. 8. HR-SEM images of Ag NPs at magnifications of (a) 5 μm, (b) 2 μm, (c) 1 μm, and (d) 500 nm.
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Scheme 1. Possible formation mechanism of silver nanoparticles using serine as a reducing agent.
Scheme 2. Graphical representation for the formation mechanism of silver microspheres.
from Ag NPs, carbonyl group of serine and PVP respectively. Metallic silver nanocrystals generally have shown an optical absorption peak approximately at 3 keV, due to the surface plasmon resonance [51]. The SAED pattern (Fig. 11) of Ag NPs prepared by microwave irradiation method showed concentric rings, which resulted from the random orientation of the crystal planes. This suggested that the sample was nano-crystalline in nature.
Fig. 12 shows the cyclic voltammogram (CV) of serine and PVP capped Ag NPs recorded at 0.05 Vs−1. Fig. 12a shows the redox peaks in the potential range of −0.6 to +1.2 V, due to the redox active components of serine. The presence of oxidation peak suggested that the components of serine might be gaining electrons from the electrode.
Fig. 10. Energy dispersive X-ray analysis of Ag NPs.
Fig. 11. SAED pattern of Ag NPs.
3.6. Cyclic voltammetry
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Fig. 12. Cyclic voltammogram (CV) of (a) pure serine and (b) PVP capped Ag NPs.
The reduction peak was due to its oxidation in the presence of an electric field. The CV of PVP capped Ag NPs (Fig. 12b) showed a well defined redox peak and was similar to that of serine. Further, additional oxidation and reduction peaks corresponding to the redox behavior of Ag0 were also observed [52,53]. The observed electrochemical activity shows that the prepared Ag NPs will have great potential in the field of electrochemical sensors [54]. 3.7. Antimicrobial activity of Ag NPs against microorganisms The synthesized Ag NP colloidal solution showed antibacterial activity against Gram-positive bacteria such as B. cereus, S. aureus, M. luteus,
Fig. 14. Bar diagram of inhibition zones of antibacterial activity of Ag NPs and AgNO3.
B. subtilis and Enterococcus and also against Gram-negative bacteria such as P. aeruginosa, S. typhi, E. coli, and K. pneumonia by showing the inhibition zones around the holes with bacteria growth on petri plates by agar diffusion method. Fig. 13 shows the result of inhibition zones of antibacterial activity. The diameters of the inhibiting zones of Ag NPs against B. cereus, S. aureus, M. luteus, B. subtilis, and Enterococcus were 19, 16, 36, 19, and 19 mm respectively for Gram-positive bacteria and P. aeruginosa, S. typhi, E. coli, and K. pneumonia were 21, 21, 21, and 12 mm respectively
Fig. 13. Results of inhibition zones of antibacterial activity (1. Ag NPs, 2. AgNO3, 3. Serine, and 4. PVP). (a) Bacillus cereus, (b) Staphylococcus aureus, (c) Pseudomonas aeruginosa, (d) Salmonella typhi, (e) Micrococcus luteus, (f) Escherichia coli, (g) Klebsiella pneumoniae, (h) Bacillus subtilis, and (i) Enterococcus.
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for Gram-negative bacteria. The diameters of the inhibiting zones of AgNO3 against B. cereus, S. aureus, M. luteus, B. subtilis and Enterococcus were 18, 14, 35, 18, 18 mm respectively for Gram-positive bacteria and P. aeruginosa, S. typhi, E. coli, and K. pneumonia were 20, 21, 20, and 13 mm respectively for Gram-negative bacteria. At the same time, the cavities containing serine and PVP respectively did not show any appreciable inhibition zones. The above given results were the mean value of the three separate experiments conducted. The inhibition zones of Ag NPs shown in the present study were found to be relatively similar, when compared with AgNO3 against Gram-positive and Gramnegative bacteria. There was a recent report [55] regarding the Ag NPs against some bacteria, which had been used by us. But, they showed lesser inhibition zone than our present synthesized Ag NPs at the same 50 μL concentration. The inhibition zone of Ag NPs against M. luteus was higher than the other bacteria. Fig. 14 shows the bar diagram of inhibition zones of Ag NPs and AgNO3. The bactericidal activity of Ag NPs is certainly due to the silver cations released from Ag NPs that act as reservoirs for the Ag+ bactericidal agent [55]. Ag+ ions are supposed to bind to sulfhydryl groups, which leads to the protein denaturation by the reduction of disulfide bonds. Also, Ag+ can form complex with the electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids. Hence, Ag NPs might have been attached to the surface of the cell membrane to disturb its function, penetrate into bacteria, and release Ag [3]. Normally several silver containing salts showed a good antimicrobial activity. But, the higher concentration of silver is harmful to both the consumers and the microbes. Hence, the smaller concentration like nano-range is much more applicable for this purpose. The present microwave-assisted synthesis of Ag NPs will be more helpful for pharmaceutical and medical applications.
[6] [7] [8] [9] [10] [11]
4. Conclusion
[35]
Ag NPs were successfully synthesized by using serine as a reducing agent and PVP as a capping agent. The hydroxyl group of serine was involved in the reduction of Ag+ ions into Ag NPs. UV–Visible, PL, FT-IR, HR-SEM, HR-TEM, EDX, SAED and CV were used to characterize the prepared Ag NPs. The synthesized Ag NPs had found to be extremely stable for more than 6 months. This present method has more advantages like cost effective, less reaction time, formation of highly stable nanoparticles, straightforward and scalable. Also, the synthesized Ag NPs could find more valuable applications in the field of sensor, medicine and environmental remedies. Acknowledgments We thank Nanotechnology Research Centre (for HR-SEM with EDX) and Interdisciplinary School of Indian System of Medicine (for FT-IR) SRM University, Chennai, India. We also heartily thank Dr. L. Devaraj Stephen for his help to record HR-TEM from IISc, Bangalore, India.
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