Ultrasonics Sonochemistry 21 (2014) 1570–1577
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Sonosynthesis of gold nanoparticles from a geranium leaf extract M. Franco-Romano a,b, M.L.A. Gil a,⇑, J.M. Palacios-Santander b, J.J. Delgado-Jaén c, I. Naranjo-Rodríguez b, J.L. Hidalgo-Hidalgo de Cisneros b, L.M. Cubillana-Aguilera b a Departamento de Química Física, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain b Departamento de Química Analítica, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain c Departamento de Ciencias de los Materiales e Ingeniería, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Metalúrgica y Química Inorgánica, Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 31 January 2014 Keywords: Gold nanoparticles Biosynthesis Ultrasound Sonocatalysis Experimental design Simplex
a b s t r a c t A rapid in situ biosynthesis of gold nanoparticles (AuNPs) is proposed in which a geranium (Pelargonium zonale) leaf extract was used as a non-toxic reducing and stabilizing agent in a sonocatalysis process based on high-power ultrasound. The synthesis process took only 3.5 min in aqueous solution under ambient conditions. The stability of the nanoparticles was studied by UV–Vis absorption spectroscopy with reference to the surface plasmon resonance (SPR) band. AuNPs have an average lifetime of about 8 weeks at 4 °C in the absence of light. The morphology and crystalline phase of the gold nanoparticles were characterized by transmission electron microscopy (TEM). The composition of the nanoparticles was evaluated by electron diffraction and X-ray energy dispersive spectroscopy (EDS). A total of 80% of the gold nanoparticles obtained in this way have a diameter in the range 8–20 nm, with an average size of 12 ± 3 nm. Fourier transform infrared spectroscopy (FTIR) indicated the presence of biomolecules that could be responsible for reducing and capping the biosynthesized gold nanoparticles. A hypothesis concerning the type of organic molecules involved in this process is also given. Experimental design linked to the simplex method was used to optimize the experimental conditions for this green synthesis route. To the best of our knowledge, this is the first time that a high-power ultrasound-based sonocatalytic process and experimental design coupled to a simplex optimization process has been used in the biosynthesis of AuNPs. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The synthesis of metal nanoparticles currently plays a fundamental role in the development of nanotechnology and, as a consequence, there is great interest in the synthesis of nanoparticles with different chemical compositions, sizes and shapes. In particular, due to their optical, magnetic, electronic, catalytic and chemical properties, gold nanoparticles (AuNPs) are employed in a range of different fields [1,2]. In recent years, the biomedical use of AuNPs, which are introduced into cells for imaging or to deliver treatments [3], requires the use of green synthesis routes based on biocompatible processes that do not involve the use of toxic chemicals. In this way, the use of biosynthetic routes that employ ⇑ Corresponding author. Address: Departamento de Química Física, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Polígono del Rio S. Pedro, S/N, 11510 Puerto Real, Cádiz, Spain. Tel.: +34 956016178; fax: +34 956016471. E-mail address: [email protected] (M.L.A. Gil). http://dx.doi.org/10.1016/j.ultsonch.2014.01.017 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
microorganisms [4–6], enzymes [7] and plants or plant extracts [8– 13] is an alternative to traditional methods for the synthesis of metal nanoparticles. The major advantage of using plants in comparison to other biological methods, such as the use of cell cultures, is that tedious preparation processes of the biological sample are not required. Among the numerous plant extracts used in the synthesis of AuNPs, such as alfalfa (Medicago Sativa) [14], Aloe Vera (Aloe saponaria) [15], Neem (Azadirachta indica) [16], chilli pepper (Capsicum annuum) [17] and others [18], geranium extract has several advantages: antioxidant activity, antimicrobial properties, bacterial growth inhibition [19,20], and the long lifetime and abundance of this vegetable species, which is widely planted throughout the temperate regions of the world. In 2003, Shankar et al. [21] reported a synthesis of AuNPs from geranium leaves. This synthetic route required a significant amount of time, around 24 h, for the process to reach completion. As an alternative technique to reduce the synthesis time markedly, sonochemistry [22,23] and, more specifically, high-power
M. Franco-Romano et al. / Ultrasonics Sonochemistry 21 (2014) 1570–1577
ultrasound-based sonochemistry is a remarkable and interesting option [24]. The synthesis of nanoparticles using sonocatalysis is based on the decomposition of a solution containing the precursor and the reducing and/or stabilizing agent(s) by irradiation with ultrasound. In general, the chemical consequences of high-intensity ultrasound arise from acoustic cavitation (the formation, growth, and collapse of bubbles), which provides the primary mechanism for sonochemical effects. During cavitation, bubble collapse produces intense local heating, high pressures and very short lifetimes. These effects, when combined with extraordinarily rapid cooling, provide a unique medium for chemical reactions under extreme conditions. Thus, cavitation serves as a means to concentrate the diffuse energy of sound into a unique set of conditions under which the metal precursor compounds decompose rapidly, leading to metal atoms that, in the presence of the appropriate stabilizing agent, agglomerate to form nanoparticles [25]. In this paper we propose a rapid in situ biosynthesis of gold nanoparticles (Fig. 1) in a process that takes only 3.5 min and combines a biological method for the generation of nanoparticles with the use of high-power ultrasound under ambient conditions. To the best of our knowledge, this is the first study in which a high-power ultrasound-based sonocatalytic process has been used in the biosynthesis of AuNPs. The sonosynthesis of AuNPs is based on the decomposition of the precursor and its subsequent reduction and stabilization by an appropriate agent in a process that involves irradiation with high-power ultrasound. An aqueous extract of geranium (Pelargonium zonale) leaves was used as the reducing and stabilizing agent. The presence of tannins, phenolics, flavonoids and lignans is responsible for the antioxidant capacity of geranium, thus allowing its use for the synthesis of nanoparticles [26]. The quantity of precursor required to perform the synthesis is lower than those reported for most other procedures found in literature. For this reason, we can also control with great precision the quantity of gold nanoparticles to be synthesized, hence lowering and optimizing the cost in time, resources and labour. In the green synthesis proposed here, H2O was used as the environmentally benign solvent in all stages of preparation. As an additional benefit, the synthesis reported offers savings in material and operating requirements, since subsequent washing, waste treatment and recycling processes are not necessary. Nanoparticles obtained in
1571
this way were characterized by transmission electron microscopy (TEM) and their stability was monitored by UV–Vis absorption spectroscopy using the surface plasmon resonance (SPR) band. The composition of the nanoparticles was determined by electron diffraction and X-ray energy dispersive spectroscopy (EDS). Moreover, Fourier transform infrared spectroscopy (FTIR) was used to obtain information about the biomolecules responsible for reducing and capping the biosynthesized AuNPs. Finally, an experimental design procedure was applied to select the most significant experimental variables for the biosynthesis and the simplex method was employed to optimize the experimental conditions of this green synthesis route. To the best of our knowledge, this is the first time that this methodology has been used to optimize the green synthesis of AuNPs.
2. Experimental Potassium tetrachloroaurate(III) (KAuCl4), purchased from Sigma–Aldrich (Sigma, Steinheim, Germany), was analytical grade and was used without further purification. Solutions were prepared with nanopure water, which was obtained by passing twice-distilled water through a Milli-Q system (18 MX cm, Millipore, Bedford, MA). The synthesis of AuNPs with geranium leaf extract was carried out by sonicating the solution with a SONICATOR 4000 high-power ultrasonic generator (MISONIX, Inc., Farmingdale, NY, USA), which operates at 20 kHz and provides a maximum power of 600 W. However, the maximum output power was never more than 36 W. The instrument was equipped with a half inch diameter titanium tip and this was directly immersed in the solution. The leaf extract was obtained by sonicating an aqueous suspension of geranium leaves with a high-power SONOPLUS HD2200 Ultrasonic Homogenizer from BANDELIN (BANDELIN electronic GmbH & Co., KG, Berlin, Germany), which operates at 20 kHz. The instrument was equipped with a 13 mm diameter titanium tip, which was directly immersed in the fluid. The UV–Visible data were obtained on a Jasco V-500 series spectrophotometer (JASCO, Easton, Maryland, USA). Transmission electron microscopy (TEM) and electron diffraction studies were carried out on a JEOL JEM-2010F (Jeol, Tokyo, Japan) microscope, equipped with a field emission gun, a
Fig. 1. Schematic description of the biosynthesis process of AuNPs using a geranium leaf extract combined with high-power ultrasound.
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M. Franco-Romano et al. / Ultrasonics Sonochemistry 21 (2014) 1570–1577
scanning-transmission electron (STEM) module, a high angle annular dark field detector (HAADF) and an energy dispersive X-ray spectroscopy (EDS) microanalyzer. The microscope was operated at 200 kV and in the STEM mode a 0.5 nm probe was used. Fourier transform infrared spectroscopy was carried out using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan) with a resolution of 4 cm 1 in the region from 4000 to 650 cm 1. Studies were performed in attenuated total reflection mode (ATR). To obtain the FTIR spectra, a freshly synthesized AuNP solution was centrifuged at 11,908 rpm for 30 min and the supernatant was then removed. The AuNPs were cleaned by removing
impurities that may have remained from the extract by dispersing the nanoparticles three times in ultrapure water. Finally, samples were dried in an oven at 60 °C for 24 h. The leaf extract was prepared by taking 2 g of thoroughly washed and finely cut leaves in a 25 mL vial with 10 mL of nanopure water. The broth was sonicated for 10 min at 50% amplitude with the high-power ultrasonic homogenizer. The sonication process breaks the walls of the leaf cells and this releases the compounds into the solution. Subsequently, leaf extract was filtered through n° 2 Whatman filter paper. Bentonite was added to the resulting solution with stirring. The mixture was refrigerated for 24 h and the protein content was separated by filtering the solution through a n° 2 Whatman filter; this step prevented flocculation and allowed cleaner nanoparticles to be obtained. At this point, the geranium leaf extract was ready to use. An experimental design methodology was applied in order to identify the variables that could be considered as significant in the synthesis process. For the experimental design, 5 factors were varied between three levels. The factors employed were as follows: the concentration of the precursor (C), potassium tetrachloroaurate(III); the volume of the plant extract (V); ultrasound amplitude (A); the time at which the plant extract was added following the start of sonication (tad); and the time at which the synthesis process was stopped (tend). The following values were applied: C (mM) = 1.0, 1.5 and 2.0; V (lL) = 75, 100 and 125; A (%) = (12, 24 and 36); tad (min) = 1, 1.5 and 2; and tend (min) = 2.5, 3.5 and 4.5. The statistical treatments and the analysis of variance results were obtained using the software package StatisticaÒ 5.1 (StatSoft, Oklahoma, USA). Having identified the significant variables in the synthesis according to the p-value (
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Sonosynthesis of gold nanoparticles from a geranium leaf extract M. Franco-Romano a,b, M.L.A. Gil a,⇑, J.M. Palacios-Santander b, J.J. Delgado-Jaén c, I. Naranjo-Rodríguez b, J.L. Hidalgo-Hidalgo de Cisneros b, L.M. Cubillana-Aguilera b a Departamento de Química Física, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain b Departamento de Química Analítica, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain c Departamento de Ciencias de los Materiales e Ingeniería, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Metalúrgica y Química Inorgánica, Facultad de Ciencias, Universidad de Cádiz, Avda. República Saharaui, S/N, 11510 Puerto Real, Cádiz, Spain
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 31 January 2014 Keywords: Gold nanoparticles Biosynthesis Ultrasound Sonocatalysis Experimental design Simplex
a b s t r a c t A rapid in situ biosynthesis of gold nanoparticles (AuNPs) is proposed in which a geranium (Pelargonium zonale) leaf extract was used as a non-toxic reducing and stabilizing agent in a sonocatalysis process based on high-power ultrasound. The synthesis process took only 3.5 min in aqueous solution under ambient conditions. The stability of the nanoparticles was studied by UV–Vis absorption spectroscopy with reference to the surface plasmon resonance (SPR) band. AuNPs have an average lifetime of about 8 weeks at 4 °C in the absence of light. The morphology and crystalline phase of the gold nanoparticles were characterized by transmission electron microscopy (TEM). The composition of the nanoparticles was evaluated by electron diffraction and X-ray energy dispersive spectroscopy (EDS). A total of 80% of the gold nanoparticles obtained in this way have a diameter in the range 8–20 nm, with an average size of 12 ± 3 nm. Fourier transform infrared spectroscopy (FTIR) indicated the presence of biomolecules that could be responsible for reducing and capping the biosynthesized gold nanoparticles. A hypothesis concerning the type of organic molecules involved in this process is also given. Experimental design linked to the simplex method was used to optimize the experimental conditions for this green synthesis route. To the best of our knowledge, this is the first time that a high-power ultrasound-based sonocatalytic process and experimental design coupled to a simplex optimization process has been used in the biosynthesis of AuNPs. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The synthesis of metal nanoparticles currently plays a fundamental role in the development of nanotechnology and, as a consequence, there is great interest in the synthesis of nanoparticles with different chemical compositions, sizes and shapes. In particular, due to their optical, magnetic, electronic, catalytic and chemical properties, gold nanoparticles (AuNPs) are employed in a range of different fields [1,2]. In recent years, the biomedical use of AuNPs, which are introduced into cells for imaging or to deliver treatments [3], requires the use of green synthesis routes based on biocompatible processes that do not involve the use of toxic chemicals. In this way, the use of biosynthetic routes that employ ⇑ Corresponding author. Address: Departamento de Química Física, Instituto Universitario de Investigación en Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Polígono del Rio S. Pedro, S/N, 11510 Puerto Real, Cádiz, Spain. Tel.: +34 956016178; fax: +34 956016471. E-mail address: [email protected] (M.L.A. Gil). http://dx.doi.org/10.1016/j.ultsonch.2014.01.017 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.
microorganisms [4–6], enzymes [7] and plants or plant extracts [8– 13] is an alternative to traditional methods for the synthesis of metal nanoparticles. The major advantage of using plants in comparison to other biological methods, such as the use of cell cultures, is that tedious preparation processes of the biological sample are not required. Among the numerous plant extracts used in the synthesis of AuNPs, such as alfalfa (Medicago Sativa) [14], Aloe Vera (Aloe saponaria) [15], Neem (Azadirachta indica) [16], chilli pepper (Capsicum annuum) [17] and others [18], geranium extract has several advantages: antioxidant activity, antimicrobial properties, bacterial growth inhibition [19,20], and the long lifetime and abundance of this vegetable species, which is widely planted throughout the temperate regions of the world. In 2003, Shankar et al. [21] reported a synthesis of AuNPs from geranium leaves. This synthetic route required a significant amount of time, around 24 h, for the process to reach completion. As an alternative technique to reduce the synthesis time markedly, sonochemistry [22,23] and, more specifically, high-power
M. Franco-Romano et al. / Ultrasonics Sonochemistry 21 (2014) 1570–1577
ultrasound-based sonochemistry is a remarkable and interesting option [24]. The synthesis of nanoparticles using sonocatalysis is based on the decomposition of a solution containing the precursor and the reducing and/or stabilizing agent(s) by irradiation with ultrasound. In general, the chemical consequences of high-intensity ultrasound arise from acoustic cavitation (the formation, growth, and collapse of bubbles), which provides the primary mechanism for sonochemical effects. During cavitation, bubble collapse produces intense local heating, high pressures and very short lifetimes. These effects, when combined with extraordinarily rapid cooling, provide a unique medium for chemical reactions under extreme conditions. Thus, cavitation serves as a means to concentrate the diffuse energy of sound into a unique set of conditions under which the metal precursor compounds decompose rapidly, leading to metal atoms that, in the presence of the appropriate stabilizing agent, agglomerate to form nanoparticles [25]. In this paper we propose a rapid in situ biosynthesis of gold nanoparticles (Fig. 1) in a process that takes only 3.5 min and combines a biological method for the generation of nanoparticles with the use of high-power ultrasound under ambient conditions. To the best of our knowledge, this is the first study in which a high-power ultrasound-based sonocatalytic process has been used in the biosynthesis of AuNPs. The sonosynthesis of AuNPs is based on the decomposition of the precursor and its subsequent reduction and stabilization by an appropriate agent in a process that involves irradiation with high-power ultrasound. An aqueous extract of geranium (Pelargonium zonale) leaves was used as the reducing and stabilizing agent. The presence of tannins, phenolics, flavonoids and lignans is responsible for the antioxidant capacity of geranium, thus allowing its use for the synthesis of nanoparticles [26]. The quantity of precursor required to perform the synthesis is lower than those reported for most other procedures found in literature. For this reason, we can also control with great precision the quantity of gold nanoparticles to be synthesized, hence lowering and optimizing the cost in time, resources and labour. In the green synthesis proposed here, H2O was used as the environmentally benign solvent in all stages of preparation. As an additional benefit, the synthesis reported offers savings in material and operating requirements, since subsequent washing, waste treatment and recycling processes are not necessary. Nanoparticles obtained in
1571
this way were characterized by transmission electron microscopy (TEM) and their stability was monitored by UV–Vis absorption spectroscopy using the surface plasmon resonance (SPR) band. The composition of the nanoparticles was determined by electron diffraction and X-ray energy dispersive spectroscopy (EDS). Moreover, Fourier transform infrared spectroscopy (FTIR) was used to obtain information about the biomolecules responsible for reducing and capping the biosynthesized AuNPs. Finally, an experimental design procedure was applied to select the most significant experimental variables for the biosynthesis and the simplex method was employed to optimize the experimental conditions of this green synthesis route. To the best of our knowledge, this is the first time that this methodology has been used to optimize the green synthesis of AuNPs.
2. Experimental Potassium tetrachloroaurate(III) (KAuCl4), purchased from Sigma–Aldrich (Sigma, Steinheim, Germany), was analytical grade and was used without further purification. Solutions were prepared with nanopure water, which was obtained by passing twice-distilled water through a Milli-Q system (18 MX cm, Millipore, Bedford, MA). The synthesis of AuNPs with geranium leaf extract was carried out by sonicating the solution with a SONICATOR 4000 high-power ultrasonic generator (MISONIX, Inc., Farmingdale, NY, USA), which operates at 20 kHz and provides a maximum power of 600 W. However, the maximum output power was never more than 36 W. The instrument was equipped with a half inch diameter titanium tip and this was directly immersed in the solution. The leaf extract was obtained by sonicating an aqueous suspension of geranium leaves with a high-power SONOPLUS HD2200 Ultrasonic Homogenizer from BANDELIN (BANDELIN electronic GmbH & Co., KG, Berlin, Germany), which operates at 20 kHz. The instrument was equipped with a 13 mm diameter titanium tip, which was directly immersed in the fluid. The UV–Visible data were obtained on a Jasco V-500 series spectrophotometer (JASCO, Easton, Maryland, USA). Transmission electron microscopy (TEM) and electron diffraction studies were carried out on a JEOL JEM-2010F (Jeol, Tokyo, Japan) microscope, equipped with a field emission gun, a
Fig. 1. Schematic description of the biosynthesis process of AuNPs using a geranium leaf extract combined with high-power ultrasound.
1572
M. Franco-Romano et al. / Ultrasonics Sonochemistry 21 (2014) 1570–1577
scanning-transmission electron (STEM) module, a high angle annular dark field detector (HAADF) and an energy dispersive X-ray spectroscopy (EDS) microanalyzer. The microscope was operated at 200 kV and in the STEM mode a 0.5 nm probe was used. Fourier transform infrared spectroscopy was carried out using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan) with a resolution of 4 cm 1 in the region from 4000 to 650 cm 1. Studies were performed in attenuated total reflection mode (ATR). To obtain the FTIR spectra, a freshly synthesized AuNP solution was centrifuged at 11,908 rpm for 30 min and the supernatant was then removed. The AuNPs were cleaned by removing
impurities that may have remained from the extract by dispersing the nanoparticles three times in ultrapure water. Finally, samples were dried in an oven at 60 °C for 24 h. The leaf extract was prepared by taking 2 g of thoroughly washed and finely cut leaves in a 25 mL vial with 10 mL of nanopure water. The broth was sonicated for 10 min at 50% amplitude with the high-power ultrasonic homogenizer. The sonication process breaks the walls of the leaf cells and this releases the compounds into the solution. Subsequently, leaf extract was filtered through n° 2 Whatman filter paper. Bentonite was added to the resulting solution with stirring. The mixture was refrigerated for 24 h and the protein content was separated by filtering the solution through a n° 2 Whatman filter; this step prevented flocculation and allowed cleaner nanoparticles to be obtained. At this point, the geranium leaf extract was ready to use. An experimental design methodology was applied in order to identify the variables that could be considered as significant in the synthesis process. For the experimental design, 5 factors were varied between three levels. The factors employed were as follows: the concentration of the precursor (C), potassium tetrachloroaurate(III); the volume of the plant extract (V); ultrasound amplitude (A); the time at which the plant extract was added following the start of sonication (tad); and the time at which the synthesis process was stopped (tend). The following values were applied: C (mM) = 1.0, 1.5 and 2.0; V (lL) = 75, 100 and 125; A (%) = (12, 24 and 36); tad (min) = 1, 1.5 and 2; and tend (min) = 2.5, 3.5 and 4.5. The statistical treatments and the analysis of variance results were obtained using the software package StatisticaÒ 5.1 (StatSoft, Oklahoma, USA). Having identified the significant variables in the synthesis according to the p-value (
