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Sonosynthesis of gold nanoparticles from a geranium leaf extract
Ultrasonics Sonochemistry 21 (2014) 1570–1577
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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
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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
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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
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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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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 (<0.05), these variables were used to optimize the experimental conditions of the synthetic method using the simplex method (programmed in LabView). After the optimization process, the optimal conditions for the synthesis of the AuNPs using the geranium leaf extract as the reducing agent can be summarized as follows: C = 1.5 mM; V = 130 lL; A = 8%; tad = 1 min; and tend = 3.5 min. 3. Results and discussion 3.1. Description of the synthesis
Fig. 2. Different ways to contract and reflect the figure in the modified simplex method.
The synthesis of gold nanoparticles was carried out by following the procedure described below.
Table 1 Conditions and results for the simplex optimization.
a
Vertex
C (mM)
V (ll)
A (%)
Absorbance (a.u.)
k (nm)
E (J)a
W (w)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1.00 2.00 1.50 1.50 2.00 1.50 2.00 1.50 2.00 2.00 1.50 1.50 1.50 1.50 1.50 1.50 1.50
75 75 118 89 113 138 157 106 140 157 160 118 195 176 200 160 129
12 12 12 32 25 34 15 28 19 15 15 5 11 6 16 15 8
2.99982 3.02816 3.56853 3.36426 3.51435 3.52723 2.77586 3.44599 3.66589 3.66541 3.59859 3.61406 3.66505 3.68364 3.66517 3.66529 3.66517
531 546 535 539 542 536 545 537 544 546 537 529 537 537 531 529 530
3,817 3,907 3,877 4,949 4,645 5,364 3,801 5,122 4,227 4,114 4,318 3,516 3,804 3,401 4,085 3,828 3,521
18.18 18.60 18.46 23.57 22.12 25.54 18.10 24.39 20.13 19.59 20.56 16.74 18.11 16.20 19.45 18.23 16.77
The amount of energy and its related power corresponds to a 3.5 min synthesis process.
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Initially, 1.25 mL of aqueous 1.5 mM KAuCl4 (pale yellow in colour) was placed in a cylindrical glass vessel and introduced into a water bath to avoid local heating generated by sonication. The high-power ultrasound probe was then immersed in the solution and, after sonication for 30 s, 100 lL of geranium leaf extract was added to the vessel. The colour of the solution immediately changed to blue/purple and then to eggplant and, finally, after 3.5 min of sonication, to a red wine colour; this colour indicates the formation of AuNPs. Once the gold nanoparticles had been obtained, the sample was centrifuged at 3000 rpm for 3 min in order to clean any residue remaining from the extract. The presence of AuNPs in the medium was confirmed by the surface plasmon resonance band (SPR) [27] observed by UV–Vis absorption spectroscopy. AuNPs show unique optical properties due to surface plasmon resonance, which depends on the dielectric constant of the surrounding medium. For spherical AuNPs, the characteristic SPR band is observed in the range 520–535 nm.
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3.2. Experimental design and optimization process In order to identify the experimental variables that could be considered as significant in the synthesis process, a 3(k p) fractional factorial design and Box–Behnken design were performed k represents the number of factors (variables), each with 3 different levels, and p represents the degrees of freedom (1 in our case). The five factors investigated and their values are indicated in the Section 2. Hence, according to this design, 3(5 1) = 34 = 81 experiments were carried out with three replicates each. A total of 243 different experiments were carried out. The main goal was to find the most significant variables that influence the absorbance at which the SPR band of AuNPs appears, as well as the wavelength of its corresponding absorbance maximum. Only a few applications of experimental design for the synthesis of gold nanoparticles have been reported in the literature [28,29]. When taking into account only one set of measurements (i.e., considering only 81 experiments without replicates), the main
Fig. 3. UV–Vis absorption spectra of AuNPs under ambient light and temperature over a 3 week period. From (a) day one to (d) day twenty-one.
Fig. 4. UV–Vis absorption spectra of AuNPs in the absence of light at 4 °C during 8 weeks. From (a) day one to (i) day fifty-six.
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conclusion drawn from the analysis of variance (ANOVA) was that the factors C and V were significant at p < 0.05 (linear and quadratic effects) in a positive way for both parameters: the absorbance and the wavelength of the AuNP SPR band. In general, and according to these results, the higher the concentration of the precursor and the volume of the reducing agent (plant extract), the better the synthesis process. Moreover, in the case of absorbance, the quadratic effect of both parameters was also significant, as were some minor interactions between these two factors. It should be noted that the influence of the factor V with respect to the usual absorbance of the plasmon was higher than that of factor C. The latter factor was more significant for increasing the anisotropy of the AuNPs (size distribution, shape and absorbance maximum of the SPR peak) as stated in Ref. [29]. In other words, the higher the concentration of KAuCl4, the higher the absorbance maximum wavelength, which in turn indicates a higher possibility of obtaining larger spherical nanoparticles, triangular or hexagonal (in projection) nanoparticles, or even Au nanorods. However, on completing this study it was found that the regression models obtained were not as good as expected: the determination coefficient and the adjusted coefficient for the regression model were: r2 = 0.55009 and radj = 0.43293 to predict absorbance, and r2 = 0.43721 and radj = 0.23006 to predict wavelength. For this reason, we attempted to enhance the regression models by including all of the replicates (243 experiments), with each replicate considered as a different block. The ANOVA results were similar to those obtained previously in terms of the significant factors, but the models were markedly better: r2 = 0.92295 and radj = 0.79214 to predict absorbance at p < 0.05. Once again, factors C and V were found to be significant at this level of significance (linear and quadratic effects), with some minor interactions between these two factors. Furthermore, ultrasound amplitude (A) was also considered to be significant (quadratic effect), together with its interaction with factor C. Hence, on considering all of the experiments at the same time, the synthesis process improved on increasing the volume of the reducing agent, the concentration of the precursor and the ultrasound amplitude, bearing in mind that anisotropy is favoured to some extent on increasing C, as one would expect. As a final conclusion, the experimental design shows that the variables that should be considered as significant for the synthesis of AuNPs with plant extracts are C, V and A. Thus, these three
factors were subsequently used to optimize the synthesis process by means of the simplex method. The use of the simplex method allows the optimization of the synthesis process by modifying all variables simultaneously as it is a multivariate method. A simplex is an n-dimensional figure with n + 1 vertices, where n is the number of variables to be optimized. In the case of two variables the simplex is a triangle and in the case of three variables it is a tetrahedron (as in this work). Each vertex represents different synthesis conditions. In the case of three variables, the simplex starts with four initial synthesis conditions A, B, C and D, to form a tetrahedron. Once the four AuNP syntheses corresponding to the appropriate conditions had been carried out, the absorbance of the AuNP SPR band was evaluated. The experiment that led to the lowest absorbance value was the discarded vertex and new conditions were calculated by a reflection operation. This process was repeated iteratively to rule out those conditions that provide the worst absorbance. This process identifies the synthesis conditions that lead to the best results. The simplex method also provides the ability to achieve the optimization more rapidly by other operations, such as expansion or contraction of the figure when the worst experiment fulfils certain conditions (Fig. 2). This approach is called the modified simplex method. The simplex method finishes when the absorbance value does not improve further – a situation that is mathematically stated by the condition: variance obtained < variance of the method (3.61 10 3). The variance of the method is obtained by carrying out ten different syntheses under the preliminary conditions established as follows: C = 1.5 mM; V = 150 lL; A = 30%; tad = 1 min; and tend = 3.5 min. The optimization of the three aforementioned variables (C, V and A) implied that the simplex was a tetrahedron. The conditions Table 2 Results obtained in the reproducibility study. Standard deviation (r), variance (r2) and relative standard deviation (RSD).
Average value
r r2 RSD
Maximun absorbance/a.u.
Wavelength/nm.
Daily
3 months
Daily
3 months
3.56002 0.06632 0.00440 1.86281
3.55358 0.07100 0.00510 2.00000
531.79 1.09 1.18 0.20
531.60 1.34 1.80 0.25
Fig. 5. UV–Vis absorption spectra of AuNPs after ten repetitions obtained by one experimentalist.
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Fig. 6. Typical HAADF-STEM (a, b) and HREM (c, e) micrograph of the sample. Particle size distribution (d) and Digital Diffraction Patterns of selected areas ((f) and inset of image (e)) are included. EDS spectra (g) of AuNPs is also shown.
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for the first four trials were set as shown in Table 1, with tad and tend initially fixed at 1 and 3.5 min, respectively. After 17 experiments, the maximum value for the absorbance of the AuNP plasmon resonance band under the optimal conditions (lower amount of reagents and power) was achieved using: C = 1.5 mM, V = 130 lL, and A = 8%. All of the synthesis processes were carried out under ambient conditions. To the best of our knowledge, this is one of the first times that an experimental design linked to a simplex optimization procedure has been applied to optimize the experimental conditions for the green synthesis of AuNPs.
(average absorbance) = 3.59781, r (standard deviation) = 0.02870 and r2 (variance) = 0.000741. The UV–Vis absorption spectra obtained are shown in Fig. 5 and it can be observed that the height and shape of the peaks are identical for the replicates. The reproducibility of the AuNPs biosynthesis was evaluated by a single experimentalist, who repeated the synthesis thirty times over 3 months. The wavelength corresponding to the absorbance maximum for the SPR band was further analyzed. The statistical data related to the results obtained for each measurement in the reproducibility study (considering daily and 3 monthly data) are given in Table 2.
3.3. Study of nanoparticle stability 3.5. Characterization of AuNPs The stability over time of the AuNPs in terms of cluster formation was studied by UV-spectroscopy. Freshly synthesized AuNPs were stored under two different sets of conditions. The AuNPs were stable for at least 3 weeks at ambient temperature and exposure to normal light conditions. The UV–Vis spectra recorded on the day of the synthesis and then every 7 days for 3 weeks are shown in Fig. 3. The inset shows the maximum absorption at k = 530 nm versus time after the synthesis. The SPR band is clearly visible and its maximum shifts to red over time, with this change being most marked over the first 7 days. When the AuNPs were stored in darkness at 4 °C they were stable for at least 8 weeks. The UV–Vis spectra recorded on the day of the synthesis and every 7 days for 8 weeks are shown in Fig. 4. The inset shows the maximum absorption at k = 530 nm versus time after the synthesis. It can be seen from the results that the AuNPs stored in the absence of light at 4 °C are stable for far longer, as one would expect. These conditions prevent the formation of clusters of the synthesized AuNPs. 3.4. Validation method After optimizing the biosynthesis through an experimental design linked to the simplex method, studies of repeatability and reproducibility were carried out in order to validate the results. The repeatability of the method was assessed by a single experimentalist who performed 10 repetitions of the gold nanoparticle synthesis from the geranium leaf extract. A statistical study of the SPR band absorbance values obtained from UV–Vis spectroscopy was carried out. The following results were obtained: AAV
The morphology, shape and size distributions of the AuNPs were characterized by transmission electron microscopy (TEM). The composition of the nanoparticles was confirmed by means of electron diffraction and X-ray energy dispersive spectroscopy (EDS). Typical TEM images of the AuNPs synthesized with the geranium leaf extract are shown in Fig. 6(a and b). AuNPs are spherical in most cases and significant aggregate formation did not occur. A small number of pentagonal and triangular (in projection) AuNPs and some Au nanorods were also observed. Moreover, the particle size is very homogeneous and follows a Gaussian distribution (Fig. 6d), with a size distribution between 6 and 35 nm. It should be noted that the particle size distribution was obtained using HAADF-STEM images (Fig. 6a and b) in combination with elemental analysis by EDS to ensure a reliable particle size distribution. A total of 80% of the gold nanoparticles have a diameter in the range 8–20 nm, with an average size of 12 ± 3 nm. The information retrieved from the Digital Diffraction Patterns (DDP) built from the HREM images (Fig. 6c and e) allowed us to corroborate the composition of the nanoparticles. As an example, the insets in Fig. 6e and f show two typical DDP of gold nanoparticles synthesized from geranium leaf extract. Both of these images were recorded along the [0 1 1] zone axis in the [0 1 1]. The EDS spectrum corresponding to the biosynthesized nanoparticles is shown in Fig. 6g. Peaks corresponding to Au clearly show the composition of the nanoparticles (the Cu signal corresponds to the grid on which the sample is supported). FTIR analyses were carried out as described in the Section 2 to identify the biomolecules present in geranium leaves that are responsible for reducing and capping the biosynthesized gold
Fig. 7. FTIR spectra of the geranium leaf extract.
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nanoparticles. The FTIR spectrum corresponding to the geranium leaf extract is shown in Fig. 7; bands at 688, 758, 1035, 1199, 1325, 1444, 1602, 1708, 2850, 2920 and 3203 cm 1 are observed. The IR bands at 688 and 758 cm 1 represent the absorption peaks of substituted aromatic rings. Peaks at 1035 and 1199 cm 1 correspond to ether or alcohol groups. The band at 1444 cm 1 is related to the symmetric and anti-symmetric scissors vibration of C–H bonds. The absorption band at 1602 cm 1 can be assigned to the vibration modes of C@C double bonds. The bands at 2850 and 2920 cm 1 can be assigned to the C–H stretching region. The spectrum shows an intense broad absorbance centered at 3203 cm 1 and this is characteristic of the hydroxyl functional group. The peak shape and the presence of bands at 1708 and 1325 cm 1 are characteristic of the carbonyl stretching [30–32]. From these observations it can be concluded that some watersoluble polyhydroxy biomolecules present in the geranium leaf extract are responsible for the biosynthesis of AuNPs. The presence of certain molecules, such as terpenoids, plays a role in the reduction of gold by the oxidation of aldehyde groups to carboxylic acids. The pH changes observed on going from the original KAuCl4 solution (pH 2.98) to the gold nanoparticle solution (pH 2.20) provide further support for this hypothesis. 4. Conclusions A rapid (only 3.5 min) in situ biosynthesis of gold nanoparticles using a geranium leaf extract as the non-toxic reducing and stabilizing agent has been developed. The synthesis process is based on sonocatalysis. The application of high-power ultrasound directly to a mixture of the gold precursor and the reducing agent in an aqueous medium has proven to be a viable alternative to produce stable (from 3 to 8 weeks) AuNPs (average diameter of 12 ± 3 nm) under ambient light, temperature and pressure conditions. The advantages of using this synthetic process are described. UV–Vis absorption spectroscopy was used to study the stability of the nanoparticles and to validate the synthesis method once the experimental conditions (C = 1.5 mM, V = 130 lL, and A = 8%) had been optimized through an experimental design in conjunction with the simplex method. The morphology and crystalline phase of the AuNPs were characterized by TEM and the composition of the nanoparticles was confirmed using electron diffraction and X-ray energy dispersive spectroscopy (EDS). FTIR spectroscopy provided useful information about the biomolecules responsible for reducing (polyhydroxylated molecules, such as terpeonids) and capping the biosynthesized AuNPs. To the best of our knowledge, this is the first time that a highpower ultrasound-based sonocatalytic process and experimental design coupled to the simplex optimization process have been applied in the biosynthesis of AuNPs. Acknowledgements Financial support from the Junta de Andalucía (P08-FQM-04006 and project TEP-6386) and Ministerio de Ciencia e Innovación of Spain and FEDER funds (CTQ2010-19058/BQU and Project MAT2010-16206 and Project GEOPETRA, Innpacto subprogram) are acknowledged. We would like to thank Mr. Pedro Braza-Lloret for his help in preparing the figures and Prof. Dr. Juan Antonio Poce-Fatou for programming the simplex method in LabView. References [1] G. Schmid, B. Corain, Nanoparticulated gold: syntheses, structures, electronics, and reactivities, Eur. J. Inorg. Chem. 17 (2003) 3081–3098. [2] P. Zhao, N. Li, D. Astruc, State of the art in gold nanoparticle synthesis, Coord. Chem. Rev. 257 (2013) 638–665.
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Sayadi, Hypoglycemic and antioxidant effects of leaf essential oil of Pelargonium graveolens L’Hér. in alloxan induced diabetic rats, Lipids Health Dis. 11 (2012) 81–83. [20] M. Bigos, M. Wasiela, D. Kalemba, M. Sienkiewicz, Antimicrobial activity of geranium oil against clinical strains of Staphylococcus aureus, Molecules 17 (2012) 10276–10291. [21] S.S. Shankar, A. Ahmad, R. Pasrichain, M. Sastry, Bioreduction of chloroaurate ions by geranium leaves and its endophytic fungus yields gold nanoparticles of different shapes, J. Mater. Chem. 13 (2003) 1822–1826. [22] K. Okitsu, A. Yue, S. Tanabe, H. Matsumoto, Y. Yobiko, Formation of colloidal gold nanoparticles in an ultrasonic field: control of rate of gold(III) reduction and size of formed gold particles, Langmuir 17 (2001) 7717–7720. [23] K. Okitsu, M. Ashokkumar, F. Grieser, Sonochemical synthesis of gold nanoparticles: effects of ultrasound frequency, J. Phys. Chem. B 109 (2005) 20673–20675. [24] L.M. Cubillana Aguilera, M. Franco Romano, M.L.A. Gil Montero, I. Naranjo Rodríguez, J.L. Hidalgo Hidalgo de Cisneros, J,M. Palacios Santander, New, fast and green procedure for the synthesis of gold nanoparticles based on sonocatalysis, Ultrason. Sonochem. 18 (2011) 789–794. [25] K.S. Suslick, G.J. Price, Applications of ultrasound to materials chemistry, Annu. Rev. Mater. Sci. 29 (1999) 295–326. [26] D. Söhretoglu, M.K. Sakar, S. Atasayar, S. Glu, H. Özgünes, O. Sterner, Polyphenolic constituents and antioxidant potential of Geranium stepporum, Rec. Nat. Prod. 5 (2011) 22–28. [27] S. Link, M.A. El-Sayed, Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods, J. Phys. Chem. B 103 (1999) 8410–8426. [28] A. Ayati, A. Ahmadpour, F.F. Bamoharram, M.M. Heravi, H. Rashidi, Optimization of the experimental conditions in synthesis of Au NPs using preyssler heteropolyacid based on the Taguchi robust design, Nano 7 (2012) 1250002/1–1250002/10. 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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
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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
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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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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 (<0.05), these variables were used to optimize the experimental conditions of the synthetic method using the simplex method (programmed in LabView). After the optimization process, the optimal conditions for the synthesis of the AuNPs using the geranium leaf extract as the reducing agent can be summarized as follows: C = 1.5 mM; V = 130 lL; A = 8%; tad = 1 min; and tend = 3.5 min. 3. Results and discussion 3.1. Description of the synthesis
Fig. 2. Different ways to contract and reflect the figure in the modified simplex method.
The synthesis of gold nanoparticles was carried out by following the procedure described below.
Table 1 Conditions and results for the simplex optimization.
a
Vertex
C (mM)
V (ll)
A (%)
Absorbance (a.u.)
k (nm)
E (J)a
W (w)a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1.00 2.00 1.50 1.50 2.00 1.50 2.00 1.50 2.00 2.00 1.50 1.50 1.50 1.50 1.50 1.50 1.50
75 75 118 89 113 138 157 106 140 157 160 118 195 176 200 160 129
12 12 12 32 25 34 15 28 19 15 15 5 11 6 16 15 8
2.99982 3.02816 3.56853 3.36426 3.51435 3.52723 2.77586 3.44599 3.66589 3.66541 3.59859 3.61406 3.66505 3.68364 3.66517 3.66529 3.66517
531 546 535 539 542 536 545 537 544 546 537 529 537 537 531 529 530
3,817 3,907 3,877 4,949 4,645 5,364 3,801 5,122 4,227 4,114 4,318 3,516 3,804 3,401 4,085 3,828 3,521
18.18 18.60 18.46 23.57 22.12 25.54 18.10 24.39 20.13 19.59 20.56 16.74 18.11 16.20 19.45 18.23 16.77
The amount of energy and its related power corresponds to a 3.5 min synthesis process.
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Initially, 1.25 mL of aqueous 1.5 mM KAuCl4 (pale yellow in colour) was placed in a cylindrical glass vessel and introduced into a water bath to avoid local heating generated by sonication. The high-power ultrasound probe was then immersed in the solution and, after sonication for 30 s, 100 lL of geranium leaf extract was added to the vessel. The colour of the solution immediately changed to blue/purple and then to eggplant and, finally, after 3.5 min of sonication, to a red wine colour; this colour indicates the formation of AuNPs. Once the gold nanoparticles had been obtained, the sample was centrifuged at 3000 rpm for 3 min in order to clean any residue remaining from the extract. The presence of AuNPs in the medium was confirmed by the surface plasmon resonance band (SPR) [27] observed by UV–Vis absorption spectroscopy. AuNPs show unique optical properties due to surface plasmon resonance, which depends on the dielectric constant of the surrounding medium. For spherical AuNPs, the characteristic SPR band is observed in the range 520–535 nm.
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3.2. Experimental design and optimization process In order to identify the experimental variables that could be considered as significant in the synthesis process, a 3(k p) fractional factorial design and Box–Behnken design were performed k represents the number of factors (variables), each with 3 different levels, and p represents the degrees of freedom (1 in our case). The five factors investigated and their values are indicated in the Section 2. Hence, according to this design, 3(5 1) = 34 = 81 experiments were carried out with three replicates each. A total of 243 different experiments were carried out. The main goal was to find the most significant variables that influence the absorbance at which the SPR band of AuNPs appears, as well as the wavelength of its corresponding absorbance maximum. Only a few applications of experimental design for the synthesis of gold nanoparticles have been reported in the literature [28,29]. When taking into account only one set of measurements (i.e., considering only 81 experiments without replicates), the main
Fig. 3. UV–Vis absorption spectra of AuNPs under ambient light and temperature over a 3 week period. From (a) day one to (d) day twenty-one.
Fig. 4. UV–Vis absorption spectra of AuNPs in the absence of light at 4 °C during 8 weeks. From (a) day one to (i) day fifty-six.
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conclusion drawn from the analysis of variance (ANOVA) was that the factors C and V were significant at p < 0.05 (linear and quadratic effects) in a positive way for both parameters: the absorbance and the wavelength of the AuNP SPR band. In general, and according to these results, the higher the concentration of the precursor and the volume of the reducing agent (plant extract), the better the synthesis process. Moreover, in the case of absorbance, the quadratic effect of both parameters was also significant, as were some minor interactions between these two factors. It should be noted that the influence of the factor V with respect to the usual absorbance of the plasmon was higher than that of factor C. The latter factor was more significant for increasing the anisotropy of the AuNPs (size distribution, shape and absorbance maximum of the SPR peak) as stated in Ref. [29]. In other words, the higher the concentration of KAuCl4, the higher the absorbance maximum wavelength, which in turn indicates a higher possibility of obtaining larger spherical nanoparticles, triangular or hexagonal (in projection) nanoparticles, or even Au nanorods. However, on completing this study it was found that the regression models obtained were not as good as expected: the determination coefficient and the adjusted coefficient for the regression model were: r2 = 0.55009 and radj = 0.43293 to predict absorbance, and r2 = 0.43721 and radj = 0.23006 to predict wavelength. For this reason, we attempted to enhance the regression models by including all of the replicates (243 experiments), with each replicate considered as a different block. The ANOVA results were similar to those obtained previously in terms of the significant factors, but the models were markedly better: r2 = 0.92295 and radj = 0.79214 to predict absorbance at p < 0.05. Once again, factors C and V were found to be significant at this level of significance (linear and quadratic effects), with some minor interactions between these two factors. Furthermore, ultrasound amplitude (A) was also considered to be significant (quadratic effect), together with its interaction with factor C. Hence, on considering all of the experiments at the same time, the synthesis process improved on increasing the volume of the reducing agent, the concentration of the precursor and the ultrasound amplitude, bearing in mind that anisotropy is favoured to some extent on increasing C, as one would expect. As a final conclusion, the experimental design shows that the variables that should be considered as significant for the synthesis of AuNPs with plant extracts are C, V and A. Thus, these three
factors were subsequently used to optimize the synthesis process by means of the simplex method. The use of the simplex method allows the optimization of the synthesis process by modifying all variables simultaneously as it is a multivariate method. A simplex is an n-dimensional figure with n + 1 vertices, where n is the number of variables to be optimized. In the case of two variables the simplex is a triangle and in the case of three variables it is a tetrahedron (as in this work). Each vertex represents different synthesis conditions. In the case of three variables, the simplex starts with four initial synthesis conditions A, B, C and D, to form a tetrahedron. Once the four AuNP syntheses corresponding to the appropriate conditions had been carried out, the absorbance of the AuNP SPR band was evaluated. The experiment that led to the lowest absorbance value was the discarded vertex and new conditions were calculated by a reflection operation. This process was repeated iteratively to rule out those conditions that provide the worst absorbance. This process identifies the synthesis conditions that lead to the best results. The simplex method also provides the ability to achieve the optimization more rapidly by other operations, such as expansion or contraction of the figure when the worst experiment fulfils certain conditions (Fig. 2). This approach is called the modified simplex method. The simplex method finishes when the absorbance value does not improve further – a situation that is mathematically stated by the condition: variance obtained < variance of the method (3.61 10 3). The variance of the method is obtained by carrying out ten different syntheses under the preliminary conditions established as follows: C = 1.5 mM; V = 150 lL; A = 30%; tad = 1 min; and tend = 3.5 min. The optimization of the three aforementioned variables (C, V and A) implied that the simplex was a tetrahedron. The conditions Table 2 Results obtained in the reproducibility study. Standard deviation (r), variance (r2) and relative standard deviation (RSD).
Average value
r r2 RSD
Maximun absorbance/a.u.
Wavelength/nm.
Daily
3 months
Daily
3 months
3.56002 0.06632 0.00440 1.86281
3.55358 0.07100 0.00510 2.00000
531.79 1.09 1.18 0.20
531.60 1.34 1.80 0.25
Fig. 5. UV–Vis absorption spectra of AuNPs after ten repetitions obtained by one experimentalist.
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Fig. 6. Typical HAADF-STEM (a, b) and HREM (c, e) micrograph of the sample. Particle size distribution (d) and Digital Diffraction Patterns of selected areas ((f) and inset of image (e)) are included. EDS spectra (g) of AuNPs is also shown.
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for the first four trials were set as shown in Table 1, with tad and tend initially fixed at 1 and 3.5 min, respectively. After 17 experiments, the maximum value for the absorbance of the AuNP plasmon resonance band under the optimal conditions (lower amount of reagents and power) was achieved using: C = 1.5 mM, V = 130 lL, and A = 8%. All of the synthesis processes were carried out under ambient conditions. To the best of our knowledge, this is one of the first times that an experimental design linked to a simplex optimization procedure has been applied to optimize the experimental conditions for the green synthesis of AuNPs.
(average absorbance) = 3.59781, r (standard deviation) = 0.02870 and r2 (variance) = 0.000741. The UV–Vis absorption spectra obtained are shown in Fig. 5 and it can be observed that the height and shape of the peaks are identical for the replicates. The reproducibility of the AuNPs biosynthesis was evaluated by a single experimentalist, who repeated the synthesis thirty times over 3 months. The wavelength corresponding to the absorbance maximum for the SPR band was further analyzed. The statistical data related to the results obtained for each measurement in the reproducibility study (considering daily and 3 monthly data) are given in Table 2.
3.3. Study of nanoparticle stability 3.5. Characterization of AuNPs The stability over time of the AuNPs in terms of cluster formation was studied by UV-spectroscopy. Freshly synthesized AuNPs were stored under two different sets of conditions. The AuNPs were stable for at least 3 weeks at ambient temperature and exposure to normal light conditions. The UV–Vis spectra recorded on the day of the synthesis and then every 7 days for 3 weeks are shown in Fig. 3. The inset shows the maximum absorption at k = 530 nm versus time after the synthesis. The SPR band is clearly visible and its maximum shifts to red over time, with this change being most marked over the first 7 days. When the AuNPs were stored in darkness at 4 °C they were stable for at least 8 weeks. The UV–Vis spectra recorded on the day of the synthesis and every 7 days for 8 weeks are shown in Fig. 4. The inset shows the maximum absorption at k = 530 nm versus time after the synthesis. It can be seen from the results that the AuNPs stored in the absence of light at 4 °C are stable for far longer, as one would expect. These conditions prevent the formation of clusters of the synthesized AuNPs. 3.4. Validation method After optimizing the biosynthesis through an experimental design linked to the simplex method, studies of repeatability and reproducibility were carried out in order to validate the results. The repeatability of the method was assessed by a single experimentalist who performed 10 repetitions of the gold nanoparticle synthesis from the geranium leaf extract. A statistical study of the SPR band absorbance values obtained from UV–Vis spectroscopy was carried out. The following results were obtained: AAV
The morphology, shape and size distributions of the AuNPs were characterized by transmission electron microscopy (TEM). The composition of the nanoparticles was confirmed by means of electron diffraction and X-ray energy dispersive spectroscopy (EDS). Typical TEM images of the AuNPs synthesized with the geranium leaf extract are shown in Fig. 6(a and b). AuNPs are spherical in most cases and significant aggregate formation did not occur. A small number of pentagonal and triangular (in projection) AuNPs and some Au nanorods were also observed. Moreover, the particle size is very homogeneous and follows a Gaussian distribution (Fig. 6d), with a size distribution between 6 and 35 nm. It should be noted that the particle size distribution was obtained using HAADF-STEM images (Fig. 6a and b) in combination with elemental analysis by EDS to ensure a reliable particle size distribution. A total of 80% of the gold nanoparticles have a diameter in the range 8–20 nm, with an average size of 12 ± 3 nm. The information retrieved from the Digital Diffraction Patterns (DDP) built from the HREM images (Fig. 6c and e) allowed us to corroborate the composition of the nanoparticles. As an example, the insets in Fig. 6e and f show two typical DDP of gold nanoparticles synthesized from geranium leaf extract. Both of these images were recorded along the [0 1 1] zone axis in the [0 1 1]. The EDS spectrum corresponding to the biosynthesized nanoparticles is shown in Fig. 6g. Peaks corresponding to Au clearly show the composition of the nanoparticles (the Cu signal corresponds to the grid on which the sample is supported). FTIR analyses were carried out as described in the Section 2 to identify the biomolecules present in geranium leaves that are responsible for reducing and capping the biosynthesized gold
Fig. 7. FTIR spectra of the geranium leaf extract.
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nanoparticles. The FTIR spectrum corresponding to the geranium leaf extract is shown in Fig. 7; bands at 688, 758, 1035, 1199, 1325, 1444, 1602, 1708, 2850, 2920 and 3203 cm 1 are observed. The IR bands at 688 and 758 cm 1 represent the absorption peaks of substituted aromatic rings. Peaks at 1035 and 1199 cm 1 correspond to ether or alcohol groups. The band at 1444 cm 1 is related to the symmetric and anti-symmetric scissors vibration of C–H bonds. The absorption band at 1602 cm 1 can be assigned to the vibration modes of C@C double bonds. The bands at 2850 and 2920 cm 1 can be assigned to the C–H stretching region. The spectrum shows an intense broad absorbance centered at 3203 cm 1 and this is characteristic of the hydroxyl functional group. The peak shape and the presence of bands at 1708 and 1325 cm 1 are characteristic of the carbonyl stretching [30–32]. From these observations it can be concluded that some watersoluble polyhydroxy biomolecules present in the geranium leaf extract are responsible for the biosynthesis of AuNPs. The presence of certain molecules, such as terpenoids, plays a role in the reduction of gold by the oxidation of aldehyde groups to carboxylic acids. The pH changes observed on going from the original KAuCl4 solution (pH 2.98) to the gold nanoparticle solution (pH 2.20) provide further support for this hypothesis. 4. Conclusions A rapid (only 3.5 min) in situ biosynthesis of gold nanoparticles using a geranium leaf extract as the non-toxic reducing and stabilizing agent has been developed. The synthesis process is based on sonocatalysis. The application of high-power ultrasound directly to a mixture of the gold precursor and the reducing agent in an aqueous medium has proven to be a viable alternative to produce stable (from 3 to 8 weeks) AuNPs (average diameter of 12 ± 3 nm) under ambient light, temperature and pressure conditions. The advantages of using this synthetic process are described. UV–Vis absorption spectroscopy was used to study the stability of the nanoparticles and to validate the synthesis method once the experimental conditions (C = 1.5 mM, V = 130 lL, and A = 8%) had been optimized through an experimental design in conjunction with the simplex method. The morphology and crystalline phase of the AuNPs were characterized by TEM and the composition of the nanoparticles was confirmed using electron diffraction and X-ray energy dispersive spectroscopy (EDS). FTIR spectroscopy provided useful information about the biomolecules responsible for reducing (polyhydroxylated molecules, such as terpeonids) and capping the biosynthesized AuNPs. To the best of our knowledge, this is the first time that a highpower ultrasound-based sonocatalytic process and experimental design coupled to the simplex optimization process have been applied in the biosynthesis of AuNPs. Acknowledgements Financial support from the Junta de Andalucía (P08-FQM-04006 and project TEP-6386) and Ministerio de Ciencia e Innovación of Spain and FEDER funds (CTQ2010-19058/BQU and Project MAT2010-16206 and Project GEOPETRA, Innpacto subprogram) are acknowledged. We would like to thank Mr. Pedro Braza-Lloret for his help in preparing the figures and Prof. Dr. Juan Antonio Poce-Fatou for programming the simplex method in LabView. References [1] G. Schmid, B. Corain, Nanoparticulated gold: syntheses, structures, electronics, and reactivities, Eur. J. Inorg. Chem. 17 (2003) 3081–3098. [2] P. Zhao, N. Li, D. Astruc, State of the art in gold nanoparticle synthesis, Coord. Chem. Rev. 257 (2013) 638–665.
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