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Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa
Colloids and Surfaces B: Biointerfaces 83 (2011) 42–48
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa E. Castro-Longoria a,∗ , Alfredo R. Vilchis-Nestor b , M. Avalos-Borja c,1 a b c
Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico Centro de Investigación en Química Sustentable, UAEMex. Piedras Blancas, Toluca, Estado de México, Mexico Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México (UNAM), Mexico
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Article history: Received 1 July 2010 Received in revised form 21 October 2010 Accepted 22 October 2010 Available online 30 October 2010 Keywords: Neurospora crassa Biosynthesis Nanoparticles Silver Gold
a b s t r a c t The development of production processes that can reduce the environmental impact, offer waste reduction and increase energy efficiency is an important step in the field of application of nanotechnology. In this work the filamentous fungus Neurospora crassa was screened and found to be successful for the production of mono and bimetallic Au/Ag nanoparticles (NPs). Analysis by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM) confirmed the biosynthesis of NPs by the fungus. The shape of NPs was found to be mainly spherical with average diameter of 11 nm for silver and 32 nm for gold, when the fungus was exposed to the aqueous solutions of 10−3 M of AgNO3 and HAuCl4 , respectively. EDS analysis also confirmed the formation of alloy-type Au/Ag bimetallic NPs when three different ratios of AgNO3 /HAuCl4 were used. TEM images of thin sections of N. crassa cells confirmed the intracellular formation of silver and gold NPs. The results obtained indicate that N. crassa can be a potential “nanofactory” for the synthesis of metallic NPs. The use of this organism will offer several advantages since it is considered as a non-pathogenic organism, has a fast growth rate, rapid capacity of metallic ions reduction, NPs stabilization and facile and economical biomass handling. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nanostructures, structures with at least one dimension between 1 and 100 nm, have been attracting steadily growing interest due to fascinating properties and emerging and exciting applications such as development of nanocomputers [1], biomolecular detection [2,3], catalysis [4], and optical devices [5] among others. The nanoparticles (NPs) of noble metals like silver and gold also have potential applications in various fields including biomedicine, where they can be used for drug and gene delivery systems [6] and treatment of some cancers [7]. The characteristics of nanostructures are closely dependent upon size, shape, size distribution, nature of the NPs and their surroundings [8]. Therefore, synthetic procedures are inclined mainly to control size, composition and
Abbreviations: NPs, nanoparticles; SEM, scanning electron microscope; TEM, transmission electron microscope; EDS, energy dispersive X-ray spectroscopy; HRTEM, high resolution transmission electron microscope. ∗ Corresponding author. Tel.: +52 646 175 05 00x27060; fax: +52 646 175 05 95x27052. E-mail address: [email protected] (E. Castro-Longoria). 1 On leave at IPICyT, San Luis Potosi, S.L.P., Mexico. 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.035
shape. Typical synthetic methodologies involve complex physical and chemical processes that use high temperature and high pressure, large amounts of energy and many toxic substances producing pollution to the environment [9]. Consequently, during the last decade, the biosynthesis of metal NPs has been explored as an alternative to developing environmentally benign procedures to obtain them. Hence, the focus has turned to nanobiotechnology [9]. New alternatives for the synthesis of metallic NPs are currently being explored through bacteria [10], yeast [11], fungi [12], plant biomass [13,14], live plants [15], and plant extracts [16,17]. The use of biological systems for the synthesis of NPs offers several advantages since the methods are easier to carry out and more economical than traditional ones. Filamentous fungi species are of particular interest because they are able to produce highly stable NPs, which prevent molecular aggregation even after prolonged storage, and therefore have improved longevity [18]. Several species including Verticillium sp., Cladosporium cladosporioides, Trichoderma asperellum, and some species of Aspergillus, Penicillium, and Fusarium have been successfully used for the synthesis of metallic NPs [18–25]. In this study, we explored for the first time the potential of the fungus Neurospora crassa to enlarge the scope of non-pathogenic biological systems for the biosynthesis of metallic nanomaterials.
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2. Materials and methods 2.1. Strain, culture conditions and synthesis of NPs. N. crassa, wild type strain N150 (FGSC # 9013), was routinely grown and maintained at 28 ◦ C in Erlenmeyer flasks containing Vogel’s Minimal Medium (VMM) [26], supplemented with 2% (w/v) sucrose and solidified with 1.5% (w/v) agar. Macroconidia were harvested and maintained in glycerol at 4 ◦ C until needed. To obtain biomass for biosynthesis studies 100 l of N. crassa macroconidia were inoculated in flasks containing 100 mL of potato dextrose broth (PDB). The cultures were incubated in an orbital shaker (Orbit Environ Shaker) at 28 ◦ C at 200 rpm for 24 h. After incubation, the biomass was harvested by filtration, followed by extensive washing with sterile distilled water to remove any residual growing media. For the biosynthesis experiments, the washed biomass samples were placed separately in several Erlenmeyer flasks to which were added 100 mL of 10−3 M HAuCl4 and 10−3 M AgNO3 aqueous solutions for gold and silver NPs, respectively. Different volume ratios (70:30, 50:50, 30:70 ml) of 10−3 M Au(III) and Ag(I) aqueous solutions were employed for the synthesis of bimetallic Au/Ag NPs. All the solutions were prepared with de-ionized water. The HAuCl4 and AgNO3 compounds were purchased from Sigma–Aldrich and used as received. The mixtures of biomass with metallic ions were incubated in a shaker at the same conditions mentioned previously for 24 h. Simultaneously, a culture was incubated under the same conditions with de-ionized water to use as a control. Aliquots of the reaction solutions were removed in order to monitor the bioreduction process from the typical absorptions bands of gold and silver NPs, produced by surface plasmon resonance, which were collected using an UV–vis Varian spectrophotometer, model Cary-300. After 24 h of incubation, the biomass from each sample was separated by filtration and small fragments of the mycelia were screened by confocal microscopy. Approximately 0.5 cm2 of the resulting biomass was dried at room temperature and the rest (between 10 and 15 g wet weight) was lyophilized for subsequent analysis. The filtered supernatant was analyzed by TEM. Confocal microscopy was carried out with an inverted Zeiss Laser Scanning Confocal Microscope LSM-510 META. Images were obtained using LSM-510 software (version 3.2; Carl Zeiss) and evaluated with an LSM-510 image examiner (version 3.2). 2.2. SEM-EDS analysis Fungal biomass before and after the formation of mono- and bimetallic Au/Ag NPs was examined by SEM on a JEOL 5300 equipped with an energy dispersive spectrometer (EDS). Analyzed samples were dried at room conditions for 5 days and small fragments were placed on pin stubs and then coated with carbon under vacuum. 2.3. TEM observations The characterization of mono- and bimetallic NPs synthesized by the fungus N. crassa was carried out by transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) using lyophilized biomass samples. Samples were ground in a mortar agate for 30 min and approximately 0.05 g of that material were suspended in 5 mL of de-ionized water containing 2 drops of isopropyl alcohol (2-propanol) and dispersed in a sonicator for 10 min. Ten microliters of this solution were placed in a carbon coated copper grid and after completely dry the grid was examined by TEM at 200 kV. The microscopes used for characterization of NPs were a JEOL 2010 and a FEI Tecnai F30.
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To investigate the localization of NPs within the N. crassa hyphae, we performed analysis of thin sections of the mycelium with NPs. The mycelia exposed for 24 h to the Au(III) and Ag(I) solutions were fixed for 2 h in 2% glutaraldehyde in 0.05 M sodium phosphate. After fixation, the mycelia was washed with 0.05 M sodium phosphate and subsequently subjected to dehydration with 20, 40, 60, 80, 95% ethanol (aqueous solutions) for 10 min at each concentration, followed by two rinses in absolute ethanol for 15 min for each rinse. Dehydrated samples were infiltrated in Spurr’s resin at the following proportions of resin/ethanol: 15/85, 30/70, 60/40, 90/10, followed by treatment with 100% of the resin. Samples were infiltrated for 4 h at each concentration and left overnight only at 100% of the resin. Embedding was carried out with 100% of the resin and polymerization at 60 ◦ C for 24 h. Ultrathin sections were cut using an ultramicrotome (Leica Ultracut R) and were placed on carbon coated copper grids. Unless otherwise stated, thin sections were examined by TEM without post-staining. For post-stained samples, grids were transferred to droplets of stain and rinse solutions and then dried at ambient conditions prior to TEM examination. Uranyl acetate (UA) and lead citrate (LC) were used as staining solutions and rinsing was carried out with distilled water. Samples were analyzed in a Hitachi H-7500 TEM at 100 kV. 3. Results and discussions The formation of NPs using N. crassa was found to be successful, as suggested by initial changes in color of the biomass upon exposure to precursor solutions. After 24 h of incubation with solutions of AgNO3 and HAuCl4 , the color of the biomass changed from pale yellow to brown or purple, respectively (Fig. 1A–C). Metallic NPs scatter and absorb light at certain wavelengths due to the resonant collective excitations of charge density at the interface between a conductor and an insulator, phenomena known as surface plasmon resonances. The optical response of metal nanoparticles (NPs) can be tuned by controlling their size, shape, and environment, providing a starting point for emerging research fields such as surface plasmon-based photonics or plasmonics [27]. Analysis by UV–vis spectroscopy of the supernatant (which contained small fragments of mycelia) revealed absorption peaks of 450 and 520 nm for the silver and gold surface plasmon, respectively (data not shown herein). Those peaks are similar to the reported characteristic absorption peaks of these noble-metal NPs [28–30]. Using as reference these absorption peaks, small fragments of mycelia were analyzed by confocal microscopy to determine the distribution of NPs within the fungal cells. Hyphae that were exposed to the ionic solutions emitted a strong fluorescence when scanned under confocal microscopy (Fig. 1D and E). From this initial scanning, NPs appeared to be distributed in most of the hyphal area. The fluorescence emitted by both metallic NPs was particularly strong. Thus, to obtain images without over-saturation, it was necessary to use a low percentage of laser intensity (1–2%). As previously mentioned, the change in color of the biomass after exposure to Au(III) and Ag(I) solutions was considered as the initial indication of the possible formation of NPs within the hyphae. Also, the strong fluorescence observed by confocal microscopy indicates the presence of foreign material in hyphae exposed to these ionic solutions because fluorescence was not present in hyphae used as the control. To corroborate the presence of Au and Ag in the fungus, we analyzed dried biomass by SEM-EDS. Fig. 2 shows micrographs of dry biomass exposed to AgNO3 (Fig. 2A) and HAuCl4 (Fig. 2B). EDS analysis confirmed the presence of silver (Fig. 2C) and gold (Fig. 2D). Also, EDS spectra recorded in the examined area of the mycelium, show signals from silver (Fig. 3A) and gold atoms (Fig. 3B). In addition, signals from C, O, P and S atoms were detected due to the presence of biomass.
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Fig. 1. N. crassa biomass exposed to aqueous solutions of AgNO3 and HAuCl4 . (A) culture at time zero; (B) culture after 24 h in AgNO3 ; (C) culture after 24 h in HAuCl4 ; (D) hypha after 24 h in AgNO3 scanned under confocal microscopy (Abs/Em 420/515–530 nm); (E) hypha after 24 h in HAuCl4 scanned under confocal microscopy (Abs/Em 543/574–691 nm). Scale bars: 10 m.
The results obtained by the techniques described above were useful as a quick test to corroborate the presence of the Au and Ag in the fungal biomass. However, in order to know detailed features of NPs, such as size and form, more specific analyses were necessary. First, we analyzed the supernatant after obtaining the fungal biomass by filtration. It was clearly observed that the supernatant obtained from biomass exposed to AgNO3 was turbid, while that obtained from biomass exposed to HAuCl4 was completely clear. In both cases the supernatant was examined by TEM. In the case of silver, a large amount of NPs was found (Fig. 3A), but in the case of gold no particles were found. From this analysis, it seems clear that in N. crassa the formation of silver NPs also occurs extracellularly, which offers the advantage of obtaining NPs faster and in large amounts. The formation of extracellular silver NPs has been also reported for yeast [11], several strains of Fusarium oxysporum [9,20,31,32], and C. cladosporioides [24]. To explore the formation of NPs in fungal biomass, we examined by TEM a small fraction of the extraction prepared from the lyophilized mycelia. Production of mono- and bimetallic NPs was
abundant in all cases. Shape of extracted NPs was nearly spherical or ellipsoidal in all cases, although other morphologies may be observed (Fig. 4). The inset of Fig. 4A and B shows more clearly the general structural features of individual NPs. In the case of gold, we also obtained some irregular and triangular shapes with truncated edges (Fig. 4B). From several observations, the triangular shapes appear to be very thin nanoplates because regardless of the increased contrast in the region at which they overlap, their corresponding edges are still detectable. Triangular shapes of silver and gold have been reported previously [28,31,33,34]. In fact, gold nanocrystals of various shapes and sizes, including regular nanoplates, spiral nanoplates, nanowalls, lamellar and spherical nanoagglomerates, were produced with Aspergillus niger biomass, mycelia-free spent medium, and the extract of the fungus [34]. To explore the particle size distribution, only those corresponding to a quasi-spherical shape were measured. Smaller sizes were obtained for silver with a mean diameter of 11 nm and particle range of 3–50 nm, the most abundant being those of 3–10 nm
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Fig. 2. Dry biomass of N. crassa analyzed by SEM-EDS. (A) Biomass exposed to AgNO3 ; (B) biomass exposed to HAuCl4 , inset (A and B) shows EDS analyzed area; (C) EDS map showing detection of silver; (D) EDS map showing detection of gold. Scale bars: (A) = 30 m; (B) = 20 m.
Fig. 3. EDS spectra recorded from N. crassa dried biomass after exposure to ionic solutions. (A) EDS from biomass exposed to AgNO3 , and (B) EDS from biomass exposed to HAuCl4 .
(Fig. 5A). Size range of gold NPs was 3–100 nm (Fig. 5B), with mean diameter of 32 nm. Bimetallic NPs were also obtained when exposing the fungus to different combinations of ionic solutions of silver and gold. Extracted NPs from the fungal biomass (Fig. 6A) were analyzed by EDS, and the X-ray signal for Ag (Fig. 6B) as well as for Au (Fig. 6C) were detected on the same site of the sample. This confirms the formation of Au/Ag bimetallic alloy-type NPs. Particle ranges were between 3 and 90 nm for Au/Ag 70/30 (Fig. 5C); 3 and 110 nm for Au/Ag 50/50 (Fig. 5D), and 4 and 45 nm for Au/Ag 30/70 (Fig. 5E). The mean diameters were 19, 51, and 35 nm, respectively. The study and production of bimetallic NPs are increasing and have received great attention from scientific and technological communities due to their catalytic activity and optoelectronic properties [35]. Some metallic NPs, such as those of silver and gold, also exhibit attractive optical properties which depend on several parameters such as size, composition, morphology, environment, and concentration. Interestingly, for Au–Ag bimetallic NPs, successive reduction of Au and Ag salts lead to the formation of multilayer structures displaying dramatic changes in color with increasing number of layers [36]. Although studies about the formation of bimetallic NPs using biological systems are few, it has been demonstrated that with the use of natural products these kinds of structures can be rapidly synthesized, obtaining NPs of different morphologies [37,38]. The understanding of the involved mechanisms of NPs formation by biological systems is important in nanobiotechnology research in order to determine even more reliable and reproducible methods for NPs production. Location of NPs within cells may provide insight into which compounds are involved in the reduction process. Therefore, intracellular location of NPs in N. crassa was investigated to determine the shape and size of the un-extracted particles. Thin sections of the hyphae exposed to ionic solutions of AgNO3 and HAuCl4 were examined by TEM. In the case of silver
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Fig. 4. Metallic nanoparticles obtained with N. crassa after 24 h of incubation in aqueous solutions of AgNO3 and HAuCl4 . (A) Silver nanoparticles; (B) gold nanoparticles; (C–E) gold/silver nanoparticles: 70/30, 50/50 and 30/70, respectively. Inset (A and B) and C and D nanoparticles examined under HRTEM.
Fig. 5. Size distribution of metallic nanoparticles extracted from N. crassa after 24 h of incubation in aqueous solutions of AgNO3 and HAuCl4 . (A) Silver nanoparticles, (B) gold nanoparticles, and (C–E) gold/silver nanoparticles; 70/30, 50/50 and 30/70, respectively. Note scale change.
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Fig. 6. Dark field TEM micrographs of bimetallic nanoparticles. (A) Bimetallic nanoparticles Au/Ag (50/50), inset shows EDS analyzed area; (B) EDS showing detection of Ag; (C) EDS showing detection of Au. EDS analysis (B and C) shows the presence of both metals in the same nanoparticles (arrows) and thus confirming the formation of Au/Ag bimetallic nanostructures alloy-type.
NPs, they were distributed throughout the cell area, although they were not visible at low magnification due to their small size. Nevertheless the presence of metallic material was evident from the high contrast clearly observed (Fig. 7A). A large number of silver NPs were visible inside the cell and also concentrated on the outer region of the cell wall (Fig. 7B). Apparently, accumulation occurred also in the nuclei (Fig. 7C). Gold NPs were also distributed in most of the cell and due to their greater size; they may be detected at lower magnifications (Fig. 7D). Shape and size of these NPs were similar to those obtained from the extractions, although fewer triangular shapes were detected (Fig. 7E). Sections stained with UA and LC revealed that NPs were concentrated in cytoplasm and the cytoplasmic membrane, but not in the cell wall (Fig. 7F). Fig. 7F shows clearly that NPs were also present on the surface of hyphae. The capacity to biosynthesize metallic NPs by fungi species has been investigated earlier and the ability to accumulate NPs on their surfaces has been reported [23], also it has been noted that different fungi show differences in affinity for the metal NPs [39]. It is clear that silver and gold NPs obtained by using N. crassa have a distinct size range and also they accumulate differently
within the cell. However, in none of our experiments did the fungus survived upon exposure to any of the ionic solutions employed, differing from the results reported for other fungi species [39]. As can be observed in Fig. 7, the integrity of the cells exposed to the ionic solutions seem to have been disturbed, the cytoplasm is somewhat collapsed leaving large empty spaces (Fig. 7A, D–F), as compared to the control sample that was not exposed to metallic ions. For instance, fungal cytoplasm in the control sample is distributed uniformly and cell components are easily distinguished, as can be observed in Fig. 8 where (in a small section of the cell) some mitochondria and microtubules (cytoskeleton components) were recognized. The reduction process inside the cell was carried out most likely by proteins and enzymes present in the cytoplasm and possibly by other such agent from organelles, thereby provoking cell death. As previously mentioned, in N. crassa silver and gold nanoparticles were found to accumulate differently within the fungal cell; perhaps this indicates that an optimal reduction process may be obtained with each metal depending on the nature of the biomolecules which are involved. The type of biomolecules implicated in the ion reduction for each of the metals which were studied remains to be determined. Such an understanding may lead
Fig. 7. TEM micrographs of N. crassa sections. (A) Section of a hypha showing intracellular localization of silver nanoparticles; (B) silver nanoparticles, arrow indicates accumulation in the outer region of the cell wall; (C) silver nanoparticles in a nucleus; (D) section of a hypha showing intracellular localization of gold nanoparticles; (E) amplification showing morphology of nanoparticles; (F) section post-stained with UA and LC showing localization of nanoparticles in cytoplasmic membrane, big arrow indicates cell wall, small arrow points out nanoparticles outside the cell wall.
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Acknowledgements Part of this work was supported by a SEP-CONACYT grant (CB-2006-1-61524). We thank Francisco Ruiz from CNyN-UNAM for technical assistance with TEM measurements and also LINAN (IPICyT) for providing TEM facilities for this work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Fig. 8. TEM micrograph of a small section of a N. crassa hypha. Sample post-stained with UA and LC. Arrow indicates cell wall; M, mitochondrion, Mt, microtubule.
[13] [14]
to a more efficient green process for the production of Ag and Au nanoparticles. Once the capacity to produce NPs by a biological agent is developed, as in the case of N. crassa, one of the main challenges is the control of size and shape of the NPs. Shape and size of NPs are two of the most important features controlling the physical, chemical, optical, and electronic properties of the nanoscopic materials [40–42]. Particle size is clearly affected by culture conditions such as pH and temperature [34] and particle shape is thought to be affected by the different functional groups found on the cell surface of microorganisms such as amine, carboxyl, sulfhydryl, and hydroxyl groups [34]. Although we found several shapes and sizes of NPs with the use of N. crassa, the great majority of them were nearly spherical and less than 100 nm in diameter. Therefore, this fungus may be useful to obtain metallic NPs offering advantages such as ease of culturing, fast growth rate (3–5 mm/h), and rapid capacity of ionic reduction, in addition to being a non-pathogenic species. The results obtained are the basis for future research and several experimental conditions using this fungus are currently investigated in order to understand how to control shape and size of NPs. 4. Conclusions
[15] [16]
[17] [18] [19] [20] [21] [22] [23]
[24] [25] [26] [27] [28] [29] [30] [31] [32]
The development of new “eco-friendly” synthetic methods for the production of nanostructured materials at lower cost and lower energy may lead to a wider range of applications in nanotechnology. In this work, we have reported a fast and easy method for the production of mono- and bimetallic Ag/Au nanoparticles using as a reduction agent the fungus N. crassa. Although all experimental procedures were carried out at ambient conditions and without varying other parameters such as pH, N. crassa has shown potential for intra- and extracellular synthesis of fairly monodispersed nanoparticles. Therefore, the use of this fungus for the production of metallic nanoparticles with more uniform size may be possible by modification of the reduction process conditions. We believe that the synthesis of nanomaterials with controlled characteristics using ecological methods will be achieved in a short period of time.
[33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Biosynthesis of silver, gold and bimetallic nanoparticles using the filamentous fungus Neurospora crassa E. Castro-Longoria a,∗ , Alfredo R. Vilchis-Nestor b , M. Avalos-Borja c,1 a b c
Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico Centro de Investigación en Química Sustentable, UAEMex. Piedras Blancas, Toluca, Estado de México, Mexico Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México (UNAM), Mexico
a r t i c l e
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Article history: Received 1 July 2010 Received in revised form 21 October 2010 Accepted 22 October 2010 Available online 30 October 2010 Keywords: Neurospora crassa Biosynthesis Nanoparticles Silver Gold
a b s t r a c t The development of production processes that can reduce the environmental impact, offer waste reduction and increase energy efficiency is an important step in the field of application of nanotechnology. In this work the filamentous fungus Neurospora crassa was screened and found to be successful for the production of mono and bimetallic Au/Ag nanoparticles (NPs). Analysis by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM) confirmed the biosynthesis of NPs by the fungus. The shape of NPs was found to be mainly spherical with average diameter of 11 nm for silver and 32 nm for gold, when the fungus was exposed to the aqueous solutions of 10−3 M of AgNO3 and HAuCl4 , respectively. EDS analysis also confirmed the formation of alloy-type Au/Ag bimetallic NPs when three different ratios of AgNO3 /HAuCl4 were used. TEM images of thin sections of N. crassa cells confirmed the intracellular formation of silver and gold NPs. The results obtained indicate that N. crassa can be a potential “nanofactory” for the synthesis of metallic NPs. The use of this organism will offer several advantages since it is considered as a non-pathogenic organism, has a fast growth rate, rapid capacity of metallic ions reduction, NPs stabilization and facile and economical biomass handling. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nanostructures, structures with at least one dimension between 1 and 100 nm, have been attracting steadily growing interest due to fascinating properties and emerging and exciting applications such as development of nanocomputers [1], biomolecular detection [2,3], catalysis [4], and optical devices [5] among others. The nanoparticles (NPs) of noble metals like silver and gold also have potential applications in various fields including biomedicine, where they can be used for drug and gene delivery systems [6] and treatment of some cancers [7]. The characteristics of nanostructures are closely dependent upon size, shape, size distribution, nature of the NPs and their surroundings [8]. Therefore, synthetic procedures are inclined mainly to control size, composition and
Abbreviations: NPs, nanoparticles; SEM, scanning electron microscope; TEM, transmission electron microscope; EDS, energy dispersive X-ray spectroscopy; HRTEM, high resolution transmission electron microscope. ∗ Corresponding author. Tel.: +52 646 175 05 00x27060; fax: +52 646 175 05 95x27052. E-mail address: [email protected] (E. Castro-Longoria). 1 On leave at IPICyT, San Luis Potosi, S.L.P., Mexico. 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.035
shape. Typical synthetic methodologies involve complex physical and chemical processes that use high temperature and high pressure, large amounts of energy and many toxic substances producing pollution to the environment [9]. Consequently, during the last decade, the biosynthesis of metal NPs has been explored as an alternative to developing environmentally benign procedures to obtain them. Hence, the focus has turned to nanobiotechnology [9]. New alternatives for the synthesis of metallic NPs are currently being explored through bacteria [10], yeast [11], fungi [12], plant biomass [13,14], live plants [15], and plant extracts [16,17]. The use of biological systems for the synthesis of NPs offers several advantages since the methods are easier to carry out and more economical than traditional ones. Filamentous fungi species are of particular interest because they are able to produce highly stable NPs, which prevent molecular aggregation even after prolonged storage, and therefore have improved longevity [18]. Several species including Verticillium sp., Cladosporium cladosporioides, Trichoderma asperellum, and some species of Aspergillus, Penicillium, and Fusarium have been successfully used for the synthesis of metallic NPs [18–25]. In this study, we explored for the first time the potential of the fungus Neurospora crassa to enlarge the scope of non-pathogenic biological systems for the biosynthesis of metallic nanomaterials.
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2. Materials and methods 2.1. Strain, culture conditions and synthesis of NPs. N. crassa, wild type strain N150 (FGSC # 9013), was routinely grown and maintained at 28 ◦ C in Erlenmeyer flasks containing Vogel’s Minimal Medium (VMM) [26], supplemented with 2% (w/v) sucrose and solidified with 1.5% (w/v) agar. Macroconidia were harvested and maintained in glycerol at 4 ◦ C until needed. To obtain biomass for biosynthesis studies 100 l of N. crassa macroconidia were inoculated in flasks containing 100 mL of potato dextrose broth (PDB). The cultures were incubated in an orbital shaker (Orbit Environ Shaker) at 28 ◦ C at 200 rpm for 24 h. After incubation, the biomass was harvested by filtration, followed by extensive washing with sterile distilled water to remove any residual growing media. For the biosynthesis experiments, the washed biomass samples were placed separately in several Erlenmeyer flasks to which were added 100 mL of 10−3 M HAuCl4 and 10−3 M AgNO3 aqueous solutions for gold and silver NPs, respectively. Different volume ratios (70:30, 50:50, 30:70 ml) of 10−3 M Au(III) and Ag(I) aqueous solutions were employed for the synthesis of bimetallic Au/Ag NPs. All the solutions were prepared with de-ionized water. The HAuCl4 and AgNO3 compounds were purchased from Sigma–Aldrich and used as received. The mixtures of biomass with metallic ions were incubated in a shaker at the same conditions mentioned previously for 24 h. Simultaneously, a culture was incubated under the same conditions with de-ionized water to use as a control. Aliquots of the reaction solutions were removed in order to monitor the bioreduction process from the typical absorptions bands of gold and silver NPs, produced by surface plasmon resonance, which were collected using an UV–vis Varian spectrophotometer, model Cary-300. After 24 h of incubation, the biomass from each sample was separated by filtration and small fragments of the mycelia were screened by confocal microscopy. Approximately 0.5 cm2 of the resulting biomass was dried at room temperature and the rest (between 10 and 15 g wet weight) was lyophilized for subsequent analysis. The filtered supernatant was analyzed by TEM. Confocal microscopy was carried out with an inverted Zeiss Laser Scanning Confocal Microscope LSM-510 META. Images were obtained using LSM-510 software (version 3.2; Carl Zeiss) and evaluated with an LSM-510 image examiner (version 3.2). 2.2. SEM-EDS analysis Fungal biomass before and after the formation of mono- and bimetallic Au/Ag NPs was examined by SEM on a JEOL 5300 equipped with an energy dispersive spectrometer (EDS). Analyzed samples were dried at room conditions for 5 days and small fragments were placed on pin stubs and then coated with carbon under vacuum. 2.3. TEM observations The characterization of mono- and bimetallic NPs synthesized by the fungus N. crassa was carried out by transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) using lyophilized biomass samples. Samples were ground in a mortar agate for 30 min and approximately 0.05 g of that material were suspended in 5 mL of de-ionized water containing 2 drops of isopropyl alcohol (2-propanol) and dispersed in a sonicator for 10 min. Ten microliters of this solution were placed in a carbon coated copper grid and after completely dry the grid was examined by TEM at 200 kV. The microscopes used for characterization of NPs were a JEOL 2010 and a FEI Tecnai F30.
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To investigate the localization of NPs within the N. crassa hyphae, we performed analysis of thin sections of the mycelium with NPs. The mycelia exposed for 24 h to the Au(III) and Ag(I) solutions were fixed for 2 h in 2% glutaraldehyde in 0.05 M sodium phosphate. After fixation, the mycelia was washed with 0.05 M sodium phosphate and subsequently subjected to dehydration with 20, 40, 60, 80, 95% ethanol (aqueous solutions) for 10 min at each concentration, followed by two rinses in absolute ethanol for 15 min for each rinse. Dehydrated samples were infiltrated in Spurr’s resin at the following proportions of resin/ethanol: 15/85, 30/70, 60/40, 90/10, followed by treatment with 100% of the resin. Samples were infiltrated for 4 h at each concentration and left overnight only at 100% of the resin. Embedding was carried out with 100% of the resin and polymerization at 60 ◦ C for 24 h. Ultrathin sections were cut using an ultramicrotome (Leica Ultracut R) and were placed on carbon coated copper grids. Unless otherwise stated, thin sections were examined by TEM without post-staining. For post-stained samples, grids were transferred to droplets of stain and rinse solutions and then dried at ambient conditions prior to TEM examination. Uranyl acetate (UA) and lead citrate (LC) were used as staining solutions and rinsing was carried out with distilled water. Samples were analyzed in a Hitachi H-7500 TEM at 100 kV. 3. Results and discussions The formation of NPs using N. crassa was found to be successful, as suggested by initial changes in color of the biomass upon exposure to precursor solutions. After 24 h of incubation with solutions of AgNO3 and HAuCl4 , the color of the biomass changed from pale yellow to brown or purple, respectively (Fig. 1A–C). Metallic NPs scatter and absorb light at certain wavelengths due to the resonant collective excitations of charge density at the interface between a conductor and an insulator, phenomena known as surface plasmon resonances. The optical response of metal nanoparticles (NPs) can be tuned by controlling their size, shape, and environment, providing a starting point for emerging research fields such as surface plasmon-based photonics or plasmonics [27]. Analysis by UV–vis spectroscopy of the supernatant (which contained small fragments of mycelia) revealed absorption peaks of 450 and 520 nm for the silver and gold surface plasmon, respectively (data not shown herein). Those peaks are similar to the reported characteristic absorption peaks of these noble-metal NPs [28–30]. Using as reference these absorption peaks, small fragments of mycelia were analyzed by confocal microscopy to determine the distribution of NPs within the fungal cells. Hyphae that were exposed to the ionic solutions emitted a strong fluorescence when scanned under confocal microscopy (Fig. 1D and E). From this initial scanning, NPs appeared to be distributed in most of the hyphal area. The fluorescence emitted by both metallic NPs was particularly strong. Thus, to obtain images without over-saturation, it was necessary to use a low percentage of laser intensity (1–2%). As previously mentioned, the change in color of the biomass after exposure to Au(III) and Ag(I) solutions was considered as the initial indication of the possible formation of NPs within the hyphae. Also, the strong fluorescence observed by confocal microscopy indicates the presence of foreign material in hyphae exposed to these ionic solutions because fluorescence was not present in hyphae used as the control. To corroborate the presence of Au and Ag in the fungus, we analyzed dried biomass by SEM-EDS. Fig. 2 shows micrographs of dry biomass exposed to AgNO3 (Fig. 2A) and HAuCl4 (Fig. 2B). EDS analysis confirmed the presence of silver (Fig. 2C) and gold (Fig. 2D). Also, EDS spectra recorded in the examined area of the mycelium, show signals from silver (Fig. 3A) and gold atoms (Fig. 3B). In addition, signals from C, O, P and S atoms were detected due to the presence of biomass.
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Fig. 1. N. crassa biomass exposed to aqueous solutions of AgNO3 and HAuCl4 . (A) culture at time zero; (B) culture after 24 h in AgNO3 ; (C) culture after 24 h in HAuCl4 ; (D) hypha after 24 h in AgNO3 scanned under confocal microscopy (Abs/Em 420/515–530 nm); (E) hypha after 24 h in HAuCl4 scanned under confocal microscopy (Abs/Em 543/574–691 nm). Scale bars: 10 m.
The results obtained by the techniques described above were useful as a quick test to corroborate the presence of the Au and Ag in the fungal biomass. However, in order to know detailed features of NPs, such as size and form, more specific analyses were necessary. First, we analyzed the supernatant after obtaining the fungal biomass by filtration. It was clearly observed that the supernatant obtained from biomass exposed to AgNO3 was turbid, while that obtained from biomass exposed to HAuCl4 was completely clear. In both cases the supernatant was examined by TEM. In the case of silver, a large amount of NPs was found (Fig. 3A), but in the case of gold no particles were found. From this analysis, it seems clear that in N. crassa the formation of silver NPs also occurs extracellularly, which offers the advantage of obtaining NPs faster and in large amounts. The formation of extracellular silver NPs has been also reported for yeast [11], several strains of Fusarium oxysporum [9,20,31,32], and C. cladosporioides [24]. To explore the formation of NPs in fungal biomass, we examined by TEM a small fraction of the extraction prepared from the lyophilized mycelia. Production of mono- and bimetallic NPs was
abundant in all cases. Shape of extracted NPs was nearly spherical or ellipsoidal in all cases, although other morphologies may be observed (Fig. 4). The inset of Fig. 4A and B shows more clearly the general structural features of individual NPs. In the case of gold, we also obtained some irregular and triangular shapes with truncated edges (Fig. 4B). From several observations, the triangular shapes appear to be very thin nanoplates because regardless of the increased contrast in the region at which they overlap, their corresponding edges are still detectable. Triangular shapes of silver and gold have been reported previously [28,31,33,34]. In fact, gold nanocrystals of various shapes and sizes, including regular nanoplates, spiral nanoplates, nanowalls, lamellar and spherical nanoagglomerates, were produced with Aspergillus niger biomass, mycelia-free spent medium, and the extract of the fungus [34]. To explore the particle size distribution, only those corresponding to a quasi-spherical shape were measured. Smaller sizes were obtained for silver with a mean diameter of 11 nm and particle range of 3–50 nm, the most abundant being those of 3–10 nm
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Fig. 2. Dry biomass of N. crassa analyzed by SEM-EDS. (A) Biomass exposed to AgNO3 ; (B) biomass exposed to HAuCl4 , inset (A and B) shows EDS analyzed area; (C) EDS map showing detection of silver; (D) EDS map showing detection of gold. Scale bars: (A) = 30 m; (B) = 20 m.
Fig. 3. EDS spectra recorded from N. crassa dried biomass after exposure to ionic solutions. (A) EDS from biomass exposed to AgNO3 , and (B) EDS from biomass exposed to HAuCl4 .
(Fig. 5A). Size range of gold NPs was 3–100 nm (Fig. 5B), with mean diameter of 32 nm. Bimetallic NPs were also obtained when exposing the fungus to different combinations of ionic solutions of silver and gold. Extracted NPs from the fungal biomass (Fig. 6A) were analyzed by EDS, and the X-ray signal for Ag (Fig. 6B) as well as for Au (Fig. 6C) were detected on the same site of the sample. This confirms the formation of Au/Ag bimetallic alloy-type NPs. Particle ranges were between 3 and 90 nm for Au/Ag 70/30 (Fig. 5C); 3 and 110 nm for Au/Ag 50/50 (Fig. 5D), and 4 and 45 nm for Au/Ag 30/70 (Fig. 5E). The mean diameters were 19, 51, and 35 nm, respectively. The study and production of bimetallic NPs are increasing and have received great attention from scientific and technological communities due to their catalytic activity and optoelectronic properties [35]. Some metallic NPs, such as those of silver and gold, also exhibit attractive optical properties which depend on several parameters such as size, composition, morphology, environment, and concentration. Interestingly, for Au–Ag bimetallic NPs, successive reduction of Au and Ag salts lead to the formation of multilayer structures displaying dramatic changes in color with increasing number of layers [36]. Although studies about the formation of bimetallic NPs using biological systems are few, it has been demonstrated that with the use of natural products these kinds of structures can be rapidly synthesized, obtaining NPs of different morphologies [37,38]. The understanding of the involved mechanisms of NPs formation by biological systems is important in nanobiotechnology research in order to determine even more reliable and reproducible methods for NPs production. Location of NPs within cells may provide insight into which compounds are involved in the reduction process. Therefore, intracellular location of NPs in N. crassa was investigated to determine the shape and size of the un-extracted particles. Thin sections of the hyphae exposed to ionic solutions of AgNO3 and HAuCl4 were examined by TEM. In the case of silver
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Fig. 4. Metallic nanoparticles obtained with N. crassa after 24 h of incubation in aqueous solutions of AgNO3 and HAuCl4 . (A) Silver nanoparticles; (B) gold nanoparticles; (C–E) gold/silver nanoparticles: 70/30, 50/50 and 30/70, respectively. Inset (A and B) and C and D nanoparticles examined under HRTEM.
Fig. 5. Size distribution of metallic nanoparticles extracted from N. crassa after 24 h of incubation in aqueous solutions of AgNO3 and HAuCl4 . (A) Silver nanoparticles, (B) gold nanoparticles, and (C–E) gold/silver nanoparticles; 70/30, 50/50 and 30/70, respectively. Note scale change.
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Fig. 6. Dark field TEM micrographs of bimetallic nanoparticles. (A) Bimetallic nanoparticles Au/Ag (50/50), inset shows EDS analyzed area; (B) EDS showing detection of Ag; (C) EDS showing detection of Au. EDS analysis (B and C) shows the presence of both metals in the same nanoparticles (arrows) and thus confirming the formation of Au/Ag bimetallic nanostructures alloy-type.
NPs, they were distributed throughout the cell area, although they were not visible at low magnification due to their small size. Nevertheless the presence of metallic material was evident from the high contrast clearly observed (Fig. 7A). A large number of silver NPs were visible inside the cell and also concentrated on the outer region of the cell wall (Fig. 7B). Apparently, accumulation occurred also in the nuclei (Fig. 7C). Gold NPs were also distributed in most of the cell and due to their greater size; they may be detected at lower magnifications (Fig. 7D). Shape and size of these NPs were similar to those obtained from the extractions, although fewer triangular shapes were detected (Fig. 7E). Sections stained with UA and LC revealed that NPs were concentrated in cytoplasm and the cytoplasmic membrane, but not in the cell wall (Fig. 7F). Fig. 7F shows clearly that NPs were also present on the surface of hyphae. The capacity to biosynthesize metallic NPs by fungi species has been investigated earlier and the ability to accumulate NPs on their surfaces has been reported [23], also it has been noted that different fungi show differences in affinity for the metal NPs [39]. It is clear that silver and gold NPs obtained by using N. crassa have a distinct size range and also they accumulate differently
within the cell. However, in none of our experiments did the fungus survived upon exposure to any of the ionic solutions employed, differing from the results reported for other fungi species [39]. As can be observed in Fig. 7, the integrity of the cells exposed to the ionic solutions seem to have been disturbed, the cytoplasm is somewhat collapsed leaving large empty spaces (Fig. 7A, D–F), as compared to the control sample that was not exposed to metallic ions. For instance, fungal cytoplasm in the control sample is distributed uniformly and cell components are easily distinguished, as can be observed in Fig. 8 where (in a small section of the cell) some mitochondria and microtubules (cytoskeleton components) were recognized. The reduction process inside the cell was carried out most likely by proteins and enzymes present in the cytoplasm and possibly by other such agent from organelles, thereby provoking cell death. As previously mentioned, in N. crassa silver and gold nanoparticles were found to accumulate differently within the fungal cell; perhaps this indicates that an optimal reduction process may be obtained with each metal depending on the nature of the biomolecules which are involved. The type of biomolecules implicated in the ion reduction for each of the metals which were studied remains to be determined. Such an understanding may lead
Fig. 7. TEM micrographs of N. crassa sections. (A) Section of a hypha showing intracellular localization of silver nanoparticles; (B) silver nanoparticles, arrow indicates accumulation in the outer region of the cell wall; (C) silver nanoparticles in a nucleus; (D) section of a hypha showing intracellular localization of gold nanoparticles; (E) amplification showing morphology of nanoparticles; (F) section post-stained with UA and LC showing localization of nanoparticles in cytoplasmic membrane, big arrow indicates cell wall, small arrow points out nanoparticles outside the cell wall.
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Acknowledgements Part of this work was supported by a SEP-CONACYT grant (CB-2006-1-61524). We thank Francisco Ruiz from CNyN-UNAM for technical assistance with TEM measurements and also LINAN (IPICyT) for providing TEM facilities for this work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Fig. 8. TEM micrograph of a small section of a N. crassa hypha. Sample post-stained with UA and LC. Arrow indicates cell wall; M, mitochondrion, Mt, microtubule.
[13] [14]
to a more efficient green process for the production of Ag and Au nanoparticles. Once the capacity to produce NPs by a biological agent is developed, as in the case of N. crassa, one of the main challenges is the control of size and shape of the NPs. Shape and size of NPs are two of the most important features controlling the physical, chemical, optical, and electronic properties of the nanoscopic materials [40–42]. Particle size is clearly affected by culture conditions such as pH and temperature [34] and particle shape is thought to be affected by the different functional groups found on the cell surface of microorganisms such as amine, carboxyl, sulfhydryl, and hydroxyl groups [34]. Although we found several shapes and sizes of NPs with the use of N. crassa, the great majority of them were nearly spherical and less than 100 nm in diameter. Therefore, this fungus may be useful to obtain metallic NPs offering advantages such as ease of culturing, fast growth rate (3–5 mm/h), and rapid capacity of ionic reduction, in addition to being a non-pathogenic species. The results obtained are the basis for future research and several experimental conditions using this fungus are currently investigated in order to understand how to control shape and size of NPs. 4. Conclusions
[15] [16]
[17] [18] [19] [20] [21] [22] [23]
[24] [25] [26] [27] [28] [29] [30] [31] [32]
The development of new “eco-friendly” synthetic methods for the production of nanostructured materials at lower cost and lower energy may lead to a wider range of applications in nanotechnology. In this work, we have reported a fast and easy method for the production of mono- and bimetallic Ag/Au nanoparticles using as a reduction agent the fungus N. crassa. Although all experimental procedures were carried out at ambient conditions and without varying other parameters such as pH, N. crassa has shown potential for intra- and extracellular synthesis of fairly monodispersed nanoparticles. Therefore, the use of this fungus for the production of metallic nanoparticles with more uniform size may be possible by modification of the reduction process conditions. We believe that the synthesis of nanomaterials with controlled characteristics using ecological methods will be achieved in a short period of time.
[33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
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