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Phytosynthesis of silver nanoparticles by Semecarpus anacardium L. leaf extract
Materials Letters 102–103 (2013) 5–7
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Phytosynthesis of silver nanoparticles by Semecarpus anacardium L. leaf extract D. Raju n, Sulekha Hazra, Urmil J. Mehta Plant Tissue Culture Division, CSIR—National Chemical Laboratory, Pune 411008, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 25 December 2012 Accepted 17 March 2013 Available online 25 March 2013
Green synthesis of silver nanoparticles (AgNPs) using Semecarpus anacardium L. leaf extract was studied. The reduction of silver (Ag þ ) ions was characterized by using UV–vis spectrophotometer showing formation of AgNPs within 15–20 min. A time dependent reaction showed the increase in the nanoparticles (NPs) with time. Transmission electron microscopy (TEM) analysis showed that the synthesized AgNPs varied from 10 to 25 nm and has spherical shape. The Fourier transform infrared (FTIR) analysis showed that phenols and protein were responsible for the formation of the AgNPs. The energy dispersive spectroscopy (EDAX) analysis confirms the formed NPs were of silver. The quantification of AgNPs was studied by inductive coupled plasma spectrometry (ICP-AES). The important outcome of this work can be value addition to the medicinal plants in synthesis of NPs for biomedical applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: Nanoparticles Semecarpus anacardium TEM Leaf extract Synthesis FTIR
1. Introduction An important area of research in nanotechnology is synthesis of nanoparticles (NPs) of different chemical compositions, sizes, and shapes. There is a growing need to develop environmentally NPs synthesis processes that do not use toxic chemicals in the synthesis protocol. As a result, researchers in the field of NPs synthesis have turned to biological systems [1]. NPs usually referred as particles with a size upto 100 nm [2,3]. Synthesis of NPs by chemical approaches is the most popular methods. But, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Since some noble metal NPs such as gold, silver and platinum NPs are widely applied to human contacting areas, there is a growing need to develop environment-friendly processes of NPs synthesis that do not use toxic chemicals [4]. Biological methods of NPs synthesis using microorganism [5], enzyme [6] and plant or plant extract have been suggested as possible eco-friendly alternatives to chemical and physical methods. Using plant for NPs synthesis can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell cultures [7]. Silver shows an inhibitory effect toward many bacterial strains and microorganisms commonly present in medical and industrial processes [8]. The most widely used and known applications of silver and AgNPs are in the medical industry which includes ointments and creams containing silver to prevent infection of burns and open wounds [9]. In addition, silver-
n
Corresponding author. Tel.: þ91 20 2590 2217; fax: þ 91 20 2590 2645. E-mail address: [email protected] (D. Raju).
0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2013.03.091
containing consumer products such as colloidal silver gel and silver-embedded fabrics are used in sports related equipments. Synthesis of gold NPs with different leaf extracts of Semecarpus was reported earlier [10]. Here in this paper, we report synthesis of polydispersed AgNPs, time dependent reaction, factors responsible for reduction of NPs and crystalline nature of NPs synthesized by a medicinally important tree species, as there are few reports on tree species. 2. Materials and methods Boil extract preparation: The reducing agent for AgNO3 (silver nitrate) was prepared by taking 30 g of Semecarpus leaves washed thoroughly with distilled water, made into small pieces and boiled in 100 ml of sterile milli Q water for a period of 30 min on water bath. After boiling, the solution was filtered with filter paper. UV Analysis: Five ml of 10−4 M solution of AgNO3 was taken in a test tube, to which 200 ml of boil extract was added and the volume was made up to 10 ml with AgNO3. The reaction was carried out at room temperature (RT). The colorless solution of AgNO3 changed to light yellowish in color indicating reduction of AgNO3. The NPs which were formed by Semecarpus leaf extract at RT were scanned at 0 h from 300 to 600 nm wavelengths with UV Spectrophotometer (Perkin-Elmer) using a dual beam operated at 1 nm resolution. The reaction mixtures were scanned under the same wavelengths after 1, 4, 8, 12, 24, 28, and 32 h. Transmission electron microscopy (TEM): The NPs of Ag were studied with TEM. The samples for TEM were prepared by placing a drop of respective NP's solution on carbon coated copper grid
6
D. Raju et al. / Materials Letters 102–103 (2013) 5–7
Fig. 1. (A) UV–vis spectra ranges from 300 to 600 nm of 10−4 M AgNO3 solution with boiled extract of Semecarpus leaves at 32 h reaction, (B) no change of color in control (10−4 M AgNO3 solution) while the color of AgNO3 changed to light brown color, after boiled extract was added for reduction, (C) time dependent reaction of color intensity indicating formation of AgNPs.
and allowing the samples to get evaporated on a filter paper. TEM measurements were performed on the JOEL model 1200EX instrument operated at an accelerating voltage of 120 kv. Scanning electron microscopy (SEM): The NPs were also analyzed with (SEM), which is attached with Phoenix EDAX to show that the reduced particles are of silver. The AgNPs synthesized by boil extract were coated on stub, which is made up of copper, zinc and other metals. Fourier transform infrared Analysis (FTIR): FTIR measurements of AgNPs were done by centrifuging the NPs at 10,000 rpm for 10 min. The supernatant was discarded, the pellet was washed with deionized water to remove the excess amount of capping agent and other compounds which are not bound to NPs and then dispensed in HPLC grade Chloroform. The particles were coated on NaCl cell substrate and scanned on Perkin-Elmer FTIR spectrum in transmittance mode at resolution of 2 cm−1. Quantification of silver by inductive coupled plasma spectrometry (ICP-AES): The unreacted silver ions in triplicates were taken and separated by centrifuging 3 ml of AgNPs solutions at 15,000 rpm for 30 min. These separated AgNPs were redispersed in 3 ml of deionized water, digested with 6 ml of aqua regia (3:1 v/v
concentrated HCl and concentrated HNO3) and the volume was made to 10 ml.
3. Results and discussion For the reduction of silver, boil extract of Semecarpus leaves was used. UV scan at 0 h reaction shows no formation of peak at 300– 600 nm. After a period of 1 h a peak was observed at 425 nm, reaching maximum intensity at 32 h. (Fig. 1A). The absorbance of UV at 425 nm was in accordance with the report [11]. A solution of 10-4 M AgNO3 was kept as control where there was no change in color formation (Fig. 1B). After a period of 15–20 min of reaction the color of the solution changed to light brownish color at RT due to plasma resonance, which indicates the formation of AgNPs (Fig. 1B). Kinetic studies were carried out to understand the increase in NPs formation. It has been observed that the intensity of reaction increases with time (Fig. 1C). Increase in intensity of the peak with an increase in time has also been reported [4]. The biomolecules were capable to reduce the silver ions up to 32 h which shows the possibility of high conversion of ionic form silver
D. Raju et al. / Materials Letters 102–103 (2013) 5–7
7
Fig. 2. (A) TEM image of AgNPs synthesized by reducing 10−4 M AgNO3 ions using boiled extract of Semecarpus leaves, (B) EDAX of AgNPs, (C) FTIR spectra recorded from AgNPs solution on NaCl cell.
to metallic nano form. The AgNPs were also quantified by using ICP-AES. The amount of AgNPs formed was 14.2 ppm. The TEM images of AgNPs synthesized by Semecarpus leaf boil extract shows that the particles are monodispersed and circular in shape. The particle size ranged from 10 to 20 nm (Fig. 2A). Fig. 2B shows EDAX recorded for boiled extract-reduced AgNPs with a strong peak of Ag. The confirmation of AgNPs by EDAX was also done by Song et al. [4]. The other peaks of Cu, Zn and a weak peak of Cl were observed which might be because of the leaf extract of Semecarpus present in the reaction solution. FTIR spectra (Fig. 2C) show peaks at 1643, 2114, and 3430 cm−1. The peak at 1643 cm−1 is the stretching vibration of amide-I C═O groups of protein [12]. The peak 2114 is from C≡C Alkynes group compounds [13]. The peak 3430 cm−1 corresponds with the stretch of phenol O–H groups [13]. The mechanism of biological AgNPs synthesis is not fully understood. In neem leaf broth, it was reported that the formation of AgNPs was by reducing sugars and/ or terpenoids [7]. In Capsicum annuum L. extract the formation of AgNPs by amine groups or secondary structure of the proteins [12]. The present study suggests the role of amide-I or phenols in the reduction of Ag ions to AgNPs. 4. Conclusions Here we report rapid formation of AgNPs by a medicinally important plant at RT. This is an environment-friendly method of
biological production of AgNPs. There are few reports on trees species and medicinally important plants. This study adds a medicinally important plant leaf extract in the formation of AgNPs.
Acknowledgments Raju D. thanks Council of Scientific and Industrial Research (CSIR) for SRF fellowship. Authors are grateful to CSIR Network program P24-COR0008 for financial support and Center for Material Characterization for TEM. References [1] Ahmad A, Senapati S, Khan MI, Kumar R, Sastry M. Langmuir 2003;19:3550–3. [2] van den Wildenberg Willems. Roadmap Report on Nanoparticles. Barcelona, Spain: W&W Espana sl; 2005. [3] Simi CK, Abraham TE. Bioprocess Biosyst Eng 2007;30:73–80. [4] Song JY, Kim BS. Bioprocess Biosyst Eng 2009;32:79–84. [5] Klaus T, Joerger R, Olsson E, Granqvist CG. Proc Natl Acad Sci USA 1999;96:13611–4. [6] Willner I, Baron R, Willner B. Adv Mater 2006;18:1109–20. [7] Shankar SS, Rai A, Ahmad A, Sastry M. J Colloid Interface Sci 2004;275:496–502. [8] Jiang H, Manolache S, Wong ACL, Denes FS. J Appl Polym Sci 2004;93:1411–22. [9] Becker RO. Met Based Drugs 1999;6:297–300. [10] Raju D, Mehta UJ, Hazra S. Trees—Struct Funct 2011;25:145–51. [11] Singh C, Baboota RK, Naik PK, Singh H. Adv Mater Lett 2012;3:279–85. [12] Li S, Shen Y, Xie A, Yu X, Qiu L, Zhang L, et al. Green Chem 2007;9:852–8. [13] 〈http://wwwchem.csustan.edu/Tutorials/INFRARED.HTM〉.
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Phytosynthesis of silver nanoparticles by Semecarpus anacardium L. leaf extract D. Raju n, Sulekha Hazra, Urmil J. Mehta Plant Tissue Culture Division, CSIR—National Chemical Laboratory, Pune 411008, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 25 December 2012 Accepted 17 March 2013 Available online 25 March 2013
Green synthesis of silver nanoparticles (AgNPs) using Semecarpus anacardium L. leaf extract was studied. The reduction of silver (Ag þ ) ions was characterized by using UV–vis spectrophotometer showing formation of AgNPs within 15–20 min. A time dependent reaction showed the increase in the nanoparticles (NPs) with time. Transmission electron microscopy (TEM) analysis showed that the synthesized AgNPs varied from 10 to 25 nm and has spherical shape. The Fourier transform infrared (FTIR) analysis showed that phenols and protein were responsible for the formation of the AgNPs. The energy dispersive spectroscopy (EDAX) analysis confirms the formed NPs were of silver. The quantification of AgNPs was studied by inductive coupled plasma spectrometry (ICP-AES). The important outcome of this work can be value addition to the medicinal plants in synthesis of NPs for biomedical applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: Nanoparticles Semecarpus anacardium TEM Leaf extract Synthesis FTIR
1. Introduction An important area of research in nanotechnology is synthesis of nanoparticles (NPs) of different chemical compositions, sizes, and shapes. There is a growing need to develop environmentally NPs synthesis processes that do not use toxic chemicals in the synthesis protocol. As a result, researchers in the field of NPs synthesis have turned to biological systems [1]. NPs usually referred as particles with a size upto 100 nm [2,3]. Synthesis of NPs by chemical approaches is the most popular methods. But, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Since some noble metal NPs such as gold, silver and platinum NPs are widely applied to human contacting areas, there is a growing need to develop environment-friendly processes of NPs synthesis that do not use toxic chemicals [4]. Biological methods of NPs synthesis using microorganism [5], enzyme [6] and plant or plant extract have been suggested as possible eco-friendly alternatives to chemical and physical methods. Using plant for NPs synthesis can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell cultures [7]. Silver shows an inhibitory effect toward many bacterial strains and microorganisms commonly present in medical and industrial processes [8]. The most widely used and known applications of silver and AgNPs are in the medical industry which includes ointments and creams containing silver to prevent infection of burns and open wounds [9]. In addition, silver-
n
Corresponding author. Tel.: þ91 20 2590 2217; fax: þ 91 20 2590 2645. E-mail address: [email protected] (D. Raju).
0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2013.03.091
containing consumer products such as colloidal silver gel and silver-embedded fabrics are used in sports related equipments. Synthesis of gold NPs with different leaf extracts of Semecarpus was reported earlier [10]. Here in this paper, we report synthesis of polydispersed AgNPs, time dependent reaction, factors responsible for reduction of NPs and crystalline nature of NPs synthesized by a medicinally important tree species, as there are few reports on tree species. 2. Materials and methods Boil extract preparation: The reducing agent for AgNO3 (silver nitrate) was prepared by taking 30 g of Semecarpus leaves washed thoroughly with distilled water, made into small pieces and boiled in 100 ml of sterile milli Q water for a period of 30 min on water bath. After boiling, the solution was filtered with filter paper. UV Analysis: Five ml of 10−4 M solution of AgNO3 was taken in a test tube, to which 200 ml of boil extract was added and the volume was made up to 10 ml with AgNO3. The reaction was carried out at room temperature (RT). The colorless solution of AgNO3 changed to light yellowish in color indicating reduction of AgNO3. The NPs which were formed by Semecarpus leaf extract at RT were scanned at 0 h from 300 to 600 nm wavelengths with UV Spectrophotometer (Perkin-Elmer) using a dual beam operated at 1 nm resolution. The reaction mixtures were scanned under the same wavelengths after 1, 4, 8, 12, 24, 28, and 32 h. Transmission electron microscopy (TEM): The NPs of Ag were studied with TEM. The samples for TEM were prepared by placing a drop of respective NP's solution on carbon coated copper grid
6
D. Raju et al. / Materials Letters 102–103 (2013) 5–7
Fig. 1. (A) UV–vis spectra ranges from 300 to 600 nm of 10−4 M AgNO3 solution with boiled extract of Semecarpus leaves at 32 h reaction, (B) no change of color in control (10−4 M AgNO3 solution) while the color of AgNO3 changed to light brown color, after boiled extract was added for reduction, (C) time dependent reaction of color intensity indicating formation of AgNPs.
and allowing the samples to get evaporated on a filter paper. TEM measurements were performed on the JOEL model 1200EX instrument operated at an accelerating voltage of 120 kv. Scanning electron microscopy (SEM): The NPs were also analyzed with (SEM), which is attached with Phoenix EDAX to show that the reduced particles are of silver. The AgNPs synthesized by boil extract were coated on stub, which is made up of copper, zinc and other metals. Fourier transform infrared Analysis (FTIR): FTIR measurements of AgNPs were done by centrifuging the NPs at 10,000 rpm for 10 min. The supernatant was discarded, the pellet was washed with deionized water to remove the excess amount of capping agent and other compounds which are not bound to NPs and then dispensed in HPLC grade Chloroform. The particles were coated on NaCl cell substrate and scanned on Perkin-Elmer FTIR spectrum in transmittance mode at resolution of 2 cm−1. Quantification of silver by inductive coupled plasma spectrometry (ICP-AES): The unreacted silver ions in triplicates were taken and separated by centrifuging 3 ml of AgNPs solutions at 15,000 rpm for 30 min. These separated AgNPs were redispersed in 3 ml of deionized water, digested with 6 ml of aqua regia (3:1 v/v
concentrated HCl and concentrated HNO3) and the volume was made to 10 ml.
3. Results and discussion For the reduction of silver, boil extract of Semecarpus leaves was used. UV scan at 0 h reaction shows no formation of peak at 300– 600 nm. After a period of 1 h a peak was observed at 425 nm, reaching maximum intensity at 32 h. (Fig. 1A). The absorbance of UV at 425 nm was in accordance with the report [11]. A solution of 10-4 M AgNO3 was kept as control where there was no change in color formation (Fig. 1B). After a period of 15–20 min of reaction the color of the solution changed to light brownish color at RT due to plasma resonance, which indicates the formation of AgNPs (Fig. 1B). Kinetic studies were carried out to understand the increase in NPs formation. It has been observed that the intensity of reaction increases with time (Fig. 1C). Increase in intensity of the peak with an increase in time has also been reported [4]. The biomolecules were capable to reduce the silver ions up to 32 h which shows the possibility of high conversion of ionic form silver
D. Raju et al. / Materials Letters 102–103 (2013) 5–7
7
Fig. 2. (A) TEM image of AgNPs synthesized by reducing 10−4 M AgNO3 ions using boiled extract of Semecarpus leaves, (B) EDAX of AgNPs, (C) FTIR spectra recorded from AgNPs solution on NaCl cell.
to metallic nano form. The AgNPs were also quantified by using ICP-AES. The amount of AgNPs formed was 14.2 ppm. The TEM images of AgNPs synthesized by Semecarpus leaf boil extract shows that the particles are monodispersed and circular in shape. The particle size ranged from 10 to 20 nm (Fig. 2A). Fig. 2B shows EDAX recorded for boiled extract-reduced AgNPs with a strong peak of Ag. The confirmation of AgNPs by EDAX was also done by Song et al. [4]. The other peaks of Cu, Zn and a weak peak of Cl were observed which might be because of the leaf extract of Semecarpus present in the reaction solution. FTIR spectra (Fig. 2C) show peaks at 1643, 2114, and 3430 cm−1. The peak at 1643 cm−1 is the stretching vibration of amide-I C═O groups of protein [12]. The peak 2114 is from C≡C Alkynes group compounds [13]. The peak 3430 cm−1 corresponds with the stretch of phenol O–H groups [13]. The mechanism of biological AgNPs synthesis is not fully understood. In neem leaf broth, it was reported that the formation of AgNPs was by reducing sugars and/ or terpenoids [7]. In Capsicum annuum L. extract the formation of AgNPs by amine groups or secondary structure of the proteins [12]. The present study suggests the role of amide-I or phenols in the reduction of Ag ions to AgNPs. 4. Conclusions Here we report rapid formation of AgNPs by a medicinally important plant at RT. This is an environment-friendly method of
biological production of AgNPs. There are few reports on trees species and medicinally important plants. This study adds a medicinally important plant leaf extract in the formation of AgNPs.
Acknowledgments Raju D. thanks Council of Scientific and Industrial Research (CSIR) for SRF fellowship. Authors are grateful to CSIR Network program P24-COR0008 for financial support and Center for Material Characterization for TEM. References [1] Ahmad A, Senapati S, Khan MI, Kumar R, Sastry M. Langmuir 2003;19:3550–3. [2] van den Wildenberg Willems. Roadmap Report on Nanoparticles. Barcelona, Spain: W&W Espana sl; 2005. [3] Simi CK, Abraham TE. Bioprocess Biosyst Eng 2007;30:73–80. [4] Song JY, Kim BS. Bioprocess Biosyst Eng 2009;32:79–84. [5] Klaus T, Joerger R, Olsson E, Granqvist CG. Proc Natl Acad Sci USA 1999;96:13611–4. [6] Willner I, Baron R, Willner B. Adv Mater 2006;18:1109–20. [7] Shankar SS, Rai A, Ahmad A, Sastry M. J Colloid Interface Sci 2004;275:496–502. [8] Jiang H, Manolache S, Wong ACL, Denes FS. J Appl Polym Sci 2004;93:1411–22. [9] Becker RO. Met Based Drugs 1999;6:297–300. [10] Raju D, Mehta UJ, Hazra S. Trees—Struct Funct 2011;25:145–51. [11] Singh C, Baboota RK, Naik PK, Singh H. Adv Mater Lett 2012;3:279–85. [12] Li S, Shen Y, Xie A, Yu X, Qiu L, Zhang L, et al. Green Chem 2007;9:852–8. [13] 〈http://wwwchem.csustan.edu/Tutorials/INFRARED.HTM〉.