Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early Online: 1–6 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2015.1041640
Microbial synthesis of Flower-shaped gold nanoparticles Priyanka Singh1, Yeon Ju Kim1, Chao Wang1, Ramya Mathiyalagan2 & Deok Chun Yang1,2 Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 05/11/15 For personal use only.
1Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Republic of Korea, and
2Graduate School of Biotechnology and Ginseng Bank, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea
2010), diagnostics, and photothermal therapy (Zhan et al. 2012). Spherical gold nanoparticles are extensively used for biomedical applications (Huang et al. 2007). The shapes and structures of nanoparticles are found to be very important factors in the modulation of their optical and physiochemical properties. The optical properties of such nanostructures are shape- and size- dependent, besides being very important and essential for applications on the biomedical platform, for instance, in photothermal therapeutics (Yuan et al. 2012) or optical cellular imaging (Yuan et al. 2013). Recently, flower-shaped (F-shaped) gold nanoparticles (also called multi-branched, urchin-like nanoparticles, and gold nanoflowers) have been proposed, which exhibit strong plasmon resonance and improve the light–matter interaction (Boca et al. 2011, Sultana et al. 2015). Considering the importance of the structure of gold nanoparticles, the present study highlights the synthesis of F-shaped gold nanoparticles using a microbial resource. Previously, the synthesis of F-shaped gold nanoparticles implied multi-step protocols, and expensive chemicals and reagents. In addition, the synthesized nanoproducts need additional stabilizing agents to remain stable for long time. Moreover, the methodologies implied the use of hazardous chemicals and solvents, which in turn required supplementary detoxification procedures in order to qualify for biological application. The biocompatibility associated with nanoparticle synthesis is the main challenge, and to produce products that can be labeled biocompatible, nanoparticles need to have a covering of polymer. Few studies have demonstrated the synthesis and application of gold nanoparticles labeled biocompatible (Winkler et al. 2011, Xie et al. 2008). Therefore, there is a growing need to develop environmentally friendly and economical techniques for the synthesis of nanoparticles without using toxic and hazardous chemicals. Microorganisms, enzymes, and plant extracts play a major role in this area of nanoparticle synthesis, and have been suggested as possible green alternatives (Vijayan et al. 2014, Singh et al. 2015a, Singh et al. 2015b, Geethalakshmi and Sarada 2012).
Abstract The shape of nanoparticles has been recognized as an important attribute that determines their applicability in various fields. The flower shape (F-shape) has been considered and is being focused on, because of its enhanced properties when compared to the properties of the spherical shape. The present study proposed the microbial synthesis of F-shaped gold nanoparticles within 48 h using the Bhargavaea indica DC1 strain. The F-shaped gold nanoparticles were synthesized extracellularly by the reduction of auric acid in the culture supernatant of B. indica DC1. The shape, size, purity, and crystalline nature of F-shaped gold nanoparticles were revealed by various instrumental techniques including UV–Vis, FE–TEM, EDX, elemental mapping, XRD, and DLS. The UV–Vis absorbance showed a maximum peak at 536 nm. FE–TEM revealed the F-shaped structure of nanoparticles. The EDX peak obtained at 2.3 keV indicated the purity. The peaks obtained on XRD analysis corresponded to the crystalline nature of the gold nanoparticles. In addition, the results of elemental mapping indicated the maximum distribution of gold elements in the nanoproduct obtained. Particle size analysis revealed that the average diameter of the F-shaped gold nanoparticles was 106 nm, with a polydispersity index (PDI) of 0.178. Thus, the methodology developed for the synthesis of F-shaped gold nanoparticles is completely green and economical. Keywords: Bhargavaea indica DC1, flower-shaped, gold nanoparticles, Microbial synthesis
Introduction In the field of nanotechnology, nanomaterials and nanoparticles are considered to be the prominent components. The commercial demand for nanoparticles is growing gradually due to their wide range of applications on biological, medical, chemical and electrical platforms. Among the many nanoparticles, gold nanoparticles have been found to have unique physiochemical properties and high biocompatibility, which are applicable in many areas, for instance, sensors, photo imaging (Ricketts et al. 2012), drug delivery (Das et al.
Correspondence: Yeon Ju Kim and Deok Chun Yang, Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Republic of Korea. Tel: 82-31-201-2100. Fax: 82-31-205-2688. E-mail: [email protected], [email protected]. (Received 27 March 2015; accepted 13 April 2015)
1
2 P. Singh et al. Keeping in mind the importance of green synthesis of gold nanoparticles, we report here the microbial synthesis of F-shaped gold nanoparticles by Bhargavaea indica DC1, which has been previously reported for the synthesis of multi-shaped silver nanoparticles (Singh et al. 2015c).
Materials and methods
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Materials
Gold (III) chloride trihydrate (HAuCl4·3H2O) was purchased from Sigma-Aldrich Chemicals, USA. All the media were purchased from Difco, MB cell, Seoul, Republic of Korea.
Isolation of bacteria The bacterial strain B. indica DC1 was isolated from fouryear-old Panax ginseng rhizospheric soil (Sodang, Republic of Korea), and has previously been reported for the synthesis of anisotropic silver nanoparticles (Singh et al. 2015c). Here, we have highlighted the synthesis of F-shaped gold nanoparticles by B. indica DC1.
Microbial synthesis of gold nanoparticles Microbial synthesis of gold nanoparticles was carried out using 24-h culture supernatant of B. indica DC1. The bacteria were grown in tryptic soy broth (TSB) and incubated for 24 h in an orbital rotatory shaker held at 37°C, at 120 rpm. Then, the culture supernatant was collected by centrifugation at 8000 rpm for 5 min, and the cell biomass was removed. The collected supernatant was further centrifuged at the same conditions to confirm that the supernatant was free from any bacterial biomass, and was further used for the microbial synthesis of gold nanoparticles. HAuCl4·3H2O at the final concentration of 1 mM was added to the culture supernatant and further incubated for 48 h in an orbital shaker, at 200 rpm and 25°C. The change in the color of the culture medium was monitored visually. For the purification and collection of nanoparticles after the incubation period, the reaction mixture was first centrifuged at 2000 rpm for 5 min to remove any components of the medium, and then centrifuged at 16 000 rpm for 10 min to collect the gold nanoparticles. The product was finally collected in pellet form after washing several times with sterile water, and was used for further characterization and confirmation (Singh et al. 2015a, Singh et al. 2015b).
and elemental mapping were prepared by dropping the pellet solution of unstained gold nanoparticles onto a carboncoated copper grid and drying in an oven at 60°C. The XRD analyses were performed on an X-ray diffractometer D8 Advance (Bruker, Germany), operated at 40 kV and 40 mA, with CuKa radiation, at a scanning rate of 6°/min, a step size of 0.02, over the 2q range of 20–80°. DLS (particle size analyzer, Photal, Otsuka Electronics, Japan) was used to monitor the size distribution profile of the biologically synthesized gold nanoparticles. The hydrodynamic diameters and PDI were analyzed at 25°C. As a reference dispersive medium, pure water, with a refractive index of 1.3328, viscosity 0.8878, and a dielectric constant of 78.3 was used. The stability of F-shaped gold nanoparticles was studied by incubating the nanoparticle reaction mixture at room temperature for different time durations. In addition, the UV–Vis absorbance was observed by adding the sodium hydroxide solution in the pH range of 4–10, to know the effect of pH on gold nanoparticles.
Results and discussion Microorganism The bacterial strain B. indica DC1, isolated from P. ginseng rhizospheric soil, was used herein to carry out the extracellular biosynthesis of F-shaped gold nanoparticles. The 16 S rRNA sequence of B. indica DC1 has been submitted to NCBI with the accession number KM819013. The strain has been deposited to the KCTC culture collection (KCTC 33595). The isolated strain has been previously reported for the biosynthesis of anisotropic silver nanoparticles (Singh et al. 2015c). The morphology of the colony after 24 h of growth on TSA has been studied and found to be circular, raised, creamcolored, of medium size, with entire margins, and odorless (Figure 1a).
Characterization of gold nanoparticles Ultraviolet–visible spectroscopy (UV–Vis), field emission transmission electron microscopy (FE–TEM), energy dispersive X-ray spectroscopy (EDX), elemental mapping (quantitation method: Cliff–Lorimer ratio), X-ray diffraction spectroscopy (XRD), and dynamic light scattering (DLS) were used to characterize the size, shape, morphology, composition, purity, and other properties of the synthesized nanoparticles. The bioreduction was monitored using a UV–Vis spectrophotometer (Ultrospec 2100 pro); this was carried out by scanning the absorbance spectra of the reaction mixture in the range of 300–800 nm. TEM, EDX, and elemental mapping were performed by using a FE–TEM (JEM-2100F, JEOL) instrument, operated at 200 kV. Samples for FE–TEM, EDX,
Figure 1. Coloy morphology of B. indica DC1 on TSA plate (a), Control flask contains TSB medium and 1 mM HAuCl4•3H2O, after incubation period (b), experimental flask contains culture supernatant of B. indica DC1 and 1 mM HAuCl4•3H2O, after incubation period.
F-shaped gold nanoparticles 3
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Synthesis and characterization of gold nanoparticles Many previous studies have reported the chemical synthesis of F-shaped gold nanoparticles (Jena and Raj 2007, Boca et al. 2011). In this study, B. indica DC1 has been used for the extracellular synthesis of F-shaped gold nanoparticles. After the incubation period, the color of the medium containing the culture supernatant of B. indica DC1 and 1 mM HAuCl4·3H2O gradually turned a faint blue within 48 h of incubation, whereas no color change was observed in the control medium without bacteria (Figure 1b, c). This faint blue could be due to the excitation of surface plasmon vibrations, which would arise from the formation of gold nanoparticles in the reaction mixture (Boca et al. 2011). The methodology is economical, ecofriendly, and avoids the use of toxic chemicals and solvents. The reaction mixture has been further scanned by UV–Vis in the range of 300–800 nm, showing a major absorbance peak at 536 nm (Figure 2). It has been reported that the band in this region corresponds to the surface plasmon resonance of gold nanoparticles (Singh et al. 2015a). Thus, the reaction mixture indicates the formation of gold nanoparticles. The F-shaped gold nanoparticles are synthesized extracellularly in the culture supernatant, which is easy to purify, and in addition, evades the downstream processing required for intracellular synthesis. Characterization of the gold nanoparticles by FE–TEM revealed that the particles were flower-like in shape and ranged in size from 30–100 nm. Figure 3a–d shows the F-shaped gold nanoparticles at 100 nm, 50 nm, 20 nm and 10 nm, respectively. The results clearly present the shape of the synthesized
Figure 2. UV-vis spectra of reaction mixture contain F-shaped gold nanoparticles.
nanoparticles, which is similar to the previously published results of studies on F-shaped gold nanoparticles (Boca et al. 2011, Sultana et al. 2015). The green synthesis of gold nanoparticles by various plants extracts and microorganisms has also been studied (Chauhan et al. 2011, Vijayan et al. 2014, Singh et al. 2015b). The biological synthesis of F-shaped gold nanoparticles using a novel strain, Talaromyces flavus, has also been studied (Priyadarshini et al. 2014). However, this is the first report of the synthesis of F-shaped gold nanoparticles by the bacterial strain B. indica DC1. The studies suggest that the floral shape of the gold nanoparticles contributes to significantly higher electrocatalytic activity and other optical
Figure 3. FE-TEM image of F-shaped gold nanoparticles at 100 nm (a), 50 nm (b), 20 nm (c) and 10 nm (c), respectively.
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4 P. Singh et al.
Figure 4. EDX spectrum of F-shaped gold nanoparticles (a), elemental mapping results indicates distribution of elements, TEM micrograph of F-shaped gold nanoparticles pellet solution (b), and F-shaped gold nanoparticles; green (c), respectively.
properties (Jena and Raj 2007). The antimicrobial property of silver nanoparticles has also been found to be increased with F-shaped silver nanoparticles, when compared to those with the spherical shape (Sahu et al. 2012). Thus, the synthesized F-shaped nanoparticles have great significance, and can be applicable in various fields. Moreover, the gold particles were synthesized without any additional reducing, stabilizing, or capping agents, thus eliminating the additional step of chemical synthesis, thereby overcoming the limitations. The mechanism behind the synthesis remains to be elucidated. Previous studies have suggested that the cell-free supernatant containing extracellular enzymes and proteins plays a major role in the reduction of HAuCl4·3H2O to gold nanoparticles and in stabilizing the synthesized product (Singh et al. 2015c). The purity of the biosynthesized gold nanoparticles was examined by EDX (Figure 4a). The EDX spectrum displayed an optical absorption band peak at 2.3 keV, which is the characteristic peak of nanosized metallic gold and corresponds to the surface plasmon resonance property of the gold nanoparticles. Due to the use of the carbon-coated copper TEM grid, the carbon and copper signals originated in the low-energy part of the spectrum. Similar results have been observed in the study of biosynthesized gold nanoparticles using ginseng root extract (Singh et al. 2015b). The results of elemental mapping of the biosynthesized F-shaped gold nanoparticles showed the distribution of elements in the electron micrograph of purified gold nanoparticles. Figure 4b corresponds to the electron micrograph of
partially purified gold nanoparticles, and Figure 4c reflects the distribution of elemental gold in the electron micrograph. The results obtained demonstrated that the deposition of gold nanoparticles was 69.92%. The distribution of elemental gold was clearly visible in the elemental maps, and was found to be the predominant element in the respective nanoproduct. Thus, the results of elemental mapping further confirmed the F-shape of the gold nanoparticles. The previous studies have demonstrated the elemental mapping results to characterize the nanoparticle distribution in the relevant nanoproducts. The results obtained here are similar to the previously reported work (Singh et al. 2015a). Figure 5 shows the X-ray
Figure 5. XRD spectrum of F-shaped gold nanoparticles.
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F-shaped gold nanoparticles 5
Figure 6. Particles size distribution of F-shaped gold nanoparticles according to volume (a), intensity (b) and number (c).
diffraction pattern of gold nanoparticles, exhibiting intense peaks in the whole spectrum of 2q value ranging from 20–80, and this pattern is similar to that of the Braggs’s reflection of gold nanocrystals. The characteristic peaks of gold nanocrystals corresponded to (111), (200), (220), and (311). The result indicates the crystalline nature of the F-shaped gold nanoparticles. The XRD results obtained are in the line of previously reported work, which displayed the similar peaks of crystalline gold nanoparticles (Jena and Raj 2007, Singh et al. 2015b). The particle size distribution profile according to volume, intensity, and number of nanoparticles shows that the size of F-shaped gold nanoparticles ranges from 50–150 nm, with an average diameter of 106 nm and a PDI 0.178 (Figure 6a–c). Thus, B. indica DC1 was demonstrated to be suitable for the simple, green, non-hazardous, economical, and eco-friendly synthesis of F-shaped gold nanoparticles without the use of any additional reducing, capping, or stabilizing agent. In the results of the stability test, there was no observable variation in the UV–Vis spectrum obtained after the incubation of reaction mixture at different time intervals, which indicates the stable nature of the F-shaped gold nanoparticles synthesized. In addition, no major change in the wavelength was detected before and after the addition of sodium hydroxide, which further confirmed the stability.
Conclusion The most important advantage of this approach includes the possibility of obtaining F-shaped gold nanoparticles using B. indica DC1. The F-shaped gold nanoparticles were synthesized by an eco-friendly and economical method, eliminating the hazardous effects of physiochemical synthesis. Moreover, the nanoparticles were synthesized without any
extra reducing and capping agents, thus eliminating the extra step in physiochemical synthesis. Furthermore, the methodology can be exploited to scale-up production at a large industrial scale, due to the ease of downstream processing. The biosynthesized F-shaped gold nanoparticles may have the potential for multifunctional use, when compared to spherical nanoparticles, for medical applications such as targeting, diagnosis, photo imaging, and drug delivery. The F-shape has additional significance for optical and electrical applications. Thus, as compared to the conventional methods, a green, time-resolved, and economically viable methodology for the synthesis of F-shaped nanoparticles was developed.
Highlights •• Microbial synthesis of F-shaped gold nanoparticles was achieved. •• The F-shaped gold nanoparticles were characterized by UV–Vis and FE–TEM. •• The nanoparticles were further characterized by EDX, elemental mapping, and XRD. •• The F-shaped gold nanoparticles were 30–100 nm in size. •• The particles were stable and monodisperse.
Acknowledgments This research was supported by the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (KIPET NO: 313038-03-2-SB010) and the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009529032014), Republic of Korea.
6 P. Singh et al.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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References Boca S, Rugina D, Pintea A, Barbu-Tudoran L, Astilean S. 2011. Flowershaped gold nanoparticles: synthesis, characterization and their application as SERS-active tags inside living cells. Nanotechnology. 22:055702. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC, et al. 2011. Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer. Int J Nanomedicine. 6:2305–2319. Das A, Mukherjee P, Singla SK, Guturu P, Frost MC, Mukhopadhyay D, et al. 2010. Fabrication and characterization of an inorganic gold and silica nanoparticle mediated drug delivery system for nitric oxide. Nanotechnology. 21:305102. Geethalakshmi R, Sarada DV. 2012. Gold and silver nanoparticles from Trianthema decandra: synthesis, characterization, and antimicrobial properties. Int J Nanomedicine. 7:5375–5384. Huang X, Jain PK, El-Sayed IH, El-Sayed MA. 2007. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine (Lond). 2:681–693. Jena BK, Raj CR. 2007. Synthesis of flower-like gold nanoparticles and their electrocatalytic activity towards the oxidation of methanol and the reduction of oxygen. Langmuir. 23:4064–4070. Priyadarshini E, Pradhan N, Sukla LB, Panda PK, Mishra BK. 2014. Biogenic synthesis of floral-shaped gold nanoparticles using a novel strain Talaromyces flavus. Ann Microbiol. 64:1055–1063. Ricketts K, Castoldi A, Guazzoni C, Ozkan C, Christodoulou C, Gibson AP, Royle GJ. 2012. A quantitative x-ray detection system for gold nanoparticle tumour biomarkers. Phys Med Biol. 57:5543–5555. Sahu SC, Mishra BK, Jena BK. 2012. Flower Shaped Silver Nanostructures: An Efficient Bacteria Exterminator. INTECH Open Access Publisher, 2012.
Singh P, Kim YJ, Wang C, Mathiyalagan R, El-Agamy Farh M, Yang DC. 2015a. Biogenic silver and gold nanoparticles synthesized using red ginseng root extract, and their applications. Artif Cells Nanomed Biotechnol. 23:1–6. Singh P, Kim YJ, Wang C, Mathiyalagan R, Yang DC. 2015b. The development of a green approach for the biosynthesis of silver and gold nanoparticles by using Panax ginseng root extract, and their biological applications. Artif Cells Nanomed Biotechnol. 14:1–8. Singh P, Kim YJ, Singh H, Wang C, Mathiyalagan R, Yang DC. 2015c. Biosynthesis of anisotropic silver nanoparticles by Bhargavaea indica and their synergistic effect with antibiotics against pathogenic microorganisms. J Nanomater. 234741. Sultana S, Djaker N, Boca-Farcau S, Salerno M, Charnaux N, Astilean S, et al. 2015. Comparative toxicity evaluation of flower-shaped and spherical gold nanoparticles on human endothelial cells. Nanotechnology. 26:055101. Vijayan SR, Santhiyagu P, Singamuthu M, Kumari Ahila N, Jayaraman R, Ethiraj K. 2014. Synthesis and characterization of silver and gold nanoparticles using aqueous extract of seaweed, Turbinaria conoides, and their antimicrofouling activity. ScientificWorldJournal. 2014:938272. Winkler K, Kaminska A, Wojciechowski T, Holyst R, Fialkowski M. 2011. Gold Micro-Flowers: One-Step Fabrication of Efficient, Highly Reproducible Surface-Enhanced Raman Spectroscopy Platform. Plasmonics. 6:697–704. Xie J, Zhang Q, Lee JY, Wang DI. 2008. The synthesis of SERS-active gold nanoflower tags for in vivo applications. ACS Nano. 2:2473–2480. Yuan H, Fales AM, Vo-Dinh T. 2012. TAT peptide-functionalized gold nanostars: enhanced intracellular delivery and efficient NIR photothermal therapy using ultralow irradiance. J Am Chem Soc. 134:11358–11361. Yuan H, Liu Y, Fales AM, Li YL, Liu J, Vo-Dinh T. 2013. Quantitative surface-enhanced resonant Raman scattering multiplexing of biocompatible gold nanostars for in vitro and ex vivo detection. Anal Chem. 85:208–212. Zhan T, Li P, Bi S, Dong B, Song H, Ren H, Wang L. 2012. 12P-conjugated PEG-modified gold nanorods combined with near-infrared laser for tumor targeting and photothermal therapy. J Nanosci Nanotechnol. 12:7198–7205.
Microbial synthesis of Flower-shaped gold nanoparticles Priyanka Singh1, Yeon Ju Kim1, Chao Wang1, Ramya Mathiyalagan2 & Deok Chun Yang1,2 Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 05/11/15 For personal use only.
1Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Republic of Korea, and
2Graduate School of Biotechnology and Ginseng Bank, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea
2010), diagnostics, and photothermal therapy (Zhan et al. 2012). Spherical gold nanoparticles are extensively used for biomedical applications (Huang et al. 2007). The shapes and structures of nanoparticles are found to be very important factors in the modulation of their optical and physiochemical properties. The optical properties of such nanostructures are shape- and size- dependent, besides being very important and essential for applications on the biomedical platform, for instance, in photothermal therapeutics (Yuan et al. 2012) or optical cellular imaging (Yuan et al. 2013). Recently, flower-shaped (F-shaped) gold nanoparticles (also called multi-branched, urchin-like nanoparticles, and gold nanoflowers) have been proposed, which exhibit strong plasmon resonance and improve the light–matter interaction (Boca et al. 2011, Sultana et al. 2015). Considering the importance of the structure of gold nanoparticles, the present study highlights the synthesis of F-shaped gold nanoparticles using a microbial resource. Previously, the synthesis of F-shaped gold nanoparticles implied multi-step protocols, and expensive chemicals and reagents. In addition, the synthesized nanoproducts need additional stabilizing agents to remain stable for long time. Moreover, the methodologies implied the use of hazardous chemicals and solvents, which in turn required supplementary detoxification procedures in order to qualify for biological application. The biocompatibility associated with nanoparticle synthesis is the main challenge, and to produce products that can be labeled biocompatible, nanoparticles need to have a covering of polymer. Few studies have demonstrated the synthesis and application of gold nanoparticles labeled biocompatible (Winkler et al. 2011, Xie et al. 2008). Therefore, there is a growing need to develop environmentally friendly and economical techniques for the synthesis of nanoparticles without using toxic and hazardous chemicals. Microorganisms, enzymes, and plant extracts play a major role in this area of nanoparticle synthesis, and have been suggested as possible green alternatives (Vijayan et al. 2014, Singh et al. 2015a, Singh et al. 2015b, Geethalakshmi and Sarada 2012).
Abstract The shape of nanoparticles has been recognized as an important attribute that determines their applicability in various fields. The flower shape (F-shape) has been considered and is being focused on, because of its enhanced properties when compared to the properties of the spherical shape. The present study proposed the microbial synthesis of F-shaped gold nanoparticles within 48 h using the Bhargavaea indica DC1 strain. The F-shaped gold nanoparticles were synthesized extracellularly by the reduction of auric acid in the culture supernatant of B. indica DC1. The shape, size, purity, and crystalline nature of F-shaped gold nanoparticles were revealed by various instrumental techniques including UV–Vis, FE–TEM, EDX, elemental mapping, XRD, and DLS. The UV–Vis absorbance showed a maximum peak at 536 nm. FE–TEM revealed the F-shaped structure of nanoparticles. The EDX peak obtained at 2.3 keV indicated the purity. The peaks obtained on XRD analysis corresponded to the crystalline nature of the gold nanoparticles. In addition, the results of elemental mapping indicated the maximum distribution of gold elements in the nanoproduct obtained. Particle size analysis revealed that the average diameter of the F-shaped gold nanoparticles was 106 nm, with a polydispersity index (PDI) of 0.178. Thus, the methodology developed for the synthesis of F-shaped gold nanoparticles is completely green and economical. Keywords: Bhargavaea indica DC1, flower-shaped, gold nanoparticles, Microbial synthesis
Introduction In the field of nanotechnology, nanomaterials and nanoparticles are considered to be the prominent components. The commercial demand for nanoparticles is growing gradually due to their wide range of applications on biological, medical, chemical and electrical platforms. Among the many nanoparticles, gold nanoparticles have been found to have unique physiochemical properties and high biocompatibility, which are applicable in many areas, for instance, sensors, photo imaging (Ricketts et al. 2012), drug delivery (Das et al.
Correspondence: Yeon Ju Kim and Deok Chun Yang, Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Republic of Korea. Tel: 82-31-201-2100. Fax: 82-31-205-2688. E-mail: [email protected], [email protected]. (Received 27 March 2015; accepted 13 April 2015)
1
2 P. Singh et al. Keeping in mind the importance of green synthesis of gold nanoparticles, we report here the microbial synthesis of F-shaped gold nanoparticles by Bhargavaea indica DC1, which has been previously reported for the synthesis of multi-shaped silver nanoparticles (Singh et al. 2015c).
Materials and methods
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Materials
Gold (III) chloride trihydrate (HAuCl4·3H2O) was purchased from Sigma-Aldrich Chemicals, USA. All the media were purchased from Difco, MB cell, Seoul, Republic of Korea.
Isolation of bacteria The bacterial strain B. indica DC1 was isolated from fouryear-old Panax ginseng rhizospheric soil (Sodang, Republic of Korea), and has previously been reported for the synthesis of anisotropic silver nanoparticles (Singh et al. 2015c). Here, we have highlighted the synthesis of F-shaped gold nanoparticles by B. indica DC1.
Microbial synthesis of gold nanoparticles Microbial synthesis of gold nanoparticles was carried out using 24-h culture supernatant of B. indica DC1. The bacteria were grown in tryptic soy broth (TSB) and incubated for 24 h in an orbital rotatory shaker held at 37°C, at 120 rpm. Then, the culture supernatant was collected by centrifugation at 8000 rpm for 5 min, and the cell biomass was removed. The collected supernatant was further centrifuged at the same conditions to confirm that the supernatant was free from any bacterial biomass, and was further used for the microbial synthesis of gold nanoparticles. HAuCl4·3H2O at the final concentration of 1 mM was added to the culture supernatant and further incubated for 48 h in an orbital shaker, at 200 rpm and 25°C. The change in the color of the culture medium was monitored visually. For the purification and collection of nanoparticles after the incubation period, the reaction mixture was first centrifuged at 2000 rpm for 5 min to remove any components of the medium, and then centrifuged at 16 000 rpm for 10 min to collect the gold nanoparticles. The product was finally collected in pellet form after washing several times with sterile water, and was used for further characterization and confirmation (Singh et al. 2015a, Singh et al. 2015b).
and elemental mapping were prepared by dropping the pellet solution of unstained gold nanoparticles onto a carboncoated copper grid and drying in an oven at 60°C. The XRD analyses were performed on an X-ray diffractometer D8 Advance (Bruker, Germany), operated at 40 kV and 40 mA, with CuKa radiation, at a scanning rate of 6°/min, a step size of 0.02, over the 2q range of 20–80°. DLS (particle size analyzer, Photal, Otsuka Electronics, Japan) was used to monitor the size distribution profile of the biologically synthesized gold nanoparticles. The hydrodynamic diameters and PDI were analyzed at 25°C. As a reference dispersive medium, pure water, with a refractive index of 1.3328, viscosity 0.8878, and a dielectric constant of 78.3 was used. The stability of F-shaped gold nanoparticles was studied by incubating the nanoparticle reaction mixture at room temperature for different time durations. In addition, the UV–Vis absorbance was observed by adding the sodium hydroxide solution in the pH range of 4–10, to know the effect of pH on gold nanoparticles.
Results and discussion Microorganism The bacterial strain B. indica DC1, isolated from P. ginseng rhizospheric soil, was used herein to carry out the extracellular biosynthesis of F-shaped gold nanoparticles. The 16 S rRNA sequence of B. indica DC1 has been submitted to NCBI with the accession number KM819013. The strain has been deposited to the KCTC culture collection (KCTC 33595). The isolated strain has been previously reported for the biosynthesis of anisotropic silver nanoparticles (Singh et al. 2015c). The morphology of the colony after 24 h of growth on TSA has been studied and found to be circular, raised, creamcolored, of medium size, with entire margins, and odorless (Figure 1a).
Characterization of gold nanoparticles Ultraviolet–visible spectroscopy (UV–Vis), field emission transmission electron microscopy (FE–TEM), energy dispersive X-ray spectroscopy (EDX), elemental mapping (quantitation method: Cliff–Lorimer ratio), X-ray diffraction spectroscopy (XRD), and dynamic light scattering (DLS) were used to characterize the size, shape, morphology, composition, purity, and other properties of the synthesized nanoparticles. The bioreduction was monitored using a UV–Vis spectrophotometer (Ultrospec 2100 pro); this was carried out by scanning the absorbance spectra of the reaction mixture in the range of 300–800 nm. TEM, EDX, and elemental mapping were performed by using a FE–TEM (JEM-2100F, JEOL) instrument, operated at 200 kV. Samples for FE–TEM, EDX,
Figure 1. Coloy morphology of B. indica DC1 on TSA plate (a), Control flask contains TSB medium and 1 mM HAuCl4•3H2O, after incubation period (b), experimental flask contains culture supernatant of B. indica DC1 and 1 mM HAuCl4•3H2O, after incubation period.
F-shaped gold nanoparticles 3
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Synthesis and characterization of gold nanoparticles Many previous studies have reported the chemical synthesis of F-shaped gold nanoparticles (Jena and Raj 2007, Boca et al. 2011). In this study, B. indica DC1 has been used for the extracellular synthesis of F-shaped gold nanoparticles. After the incubation period, the color of the medium containing the culture supernatant of B. indica DC1 and 1 mM HAuCl4·3H2O gradually turned a faint blue within 48 h of incubation, whereas no color change was observed in the control medium without bacteria (Figure 1b, c). This faint blue could be due to the excitation of surface plasmon vibrations, which would arise from the formation of gold nanoparticles in the reaction mixture (Boca et al. 2011). The methodology is economical, ecofriendly, and avoids the use of toxic chemicals and solvents. The reaction mixture has been further scanned by UV–Vis in the range of 300–800 nm, showing a major absorbance peak at 536 nm (Figure 2). It has been reported that the band in this region corresponds to the surface plasmon resonance of gold nanoparticles (Singh et al. 2015a). Thus, the reaction mixture indicates the formation of gold nanoparticles. The F-shaped gold nanoparticles are synthesized extracellularly in the culture supernatant, which is easy to purify, and in addition, evades the downstream processing required for intracellular synthesis. Characterization of the gold nanoparticles by FE–TEM revealed that the particles were flower-like in shape and ranged in size from 30–100 nm. Figure 3a–d shows the F-shaped gold nanoparticles at 100 nm, 50 nm, 20 nm and 10 nm, respectively. The results clearly present the shape of the synthesized
Figure 2. UV-vis spectra of reaction mixture contain F-shaped gold nanoparticles.
nanoparticles, which is similar to the previously published results of studies on F-shaped gold nanoparticles (Boca et al. 2011, Sultana et al. 2015). The green synthesis of gold nanoparticles by various plants extracts and microorganisms has also been studied (Chauhan et al. 2011, Vijayan et al. 2014, Singh et al. 2015b). The biological synthesis of F-shaped gold nanoparticles using a novel strain, Talaromyces flavus, has also been studied (Priyadarshini et al. 2014). However, this is the first report of the synthesis of F-shaped gold nanoparticles by the bacterial strain B. indica DC1. The studies suggest that the floral shape of the gold nanoparticles contributes to significantly higher electrocatalytic activity and other optical
Figure 3. FE-TEM image of F-shaped gold nanoparticles at 100 nm (a), 50 nm (b), 20 nm (c) and 10 nm (c), respectively.
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Figure 4. EDX spectrum of F-shaped gold nanoparticles (a), elemental mapping results indicates distribution of elements, TEM micrograph of F-shaped gold nanoparticles pellet solution (b), and F-shaped gold nanoparticles; green (c), respectively.
properties (Jena and Raj 2007). The antimicrobial property of silver nanoparticles has also been found to be increased with F-shaped silver nanoparticles, when compared to those with the spherical shape (Sahu et al. 2012). Thus, the synthesized F-shaped nanoparticles have great significance, and can be applicable in various fields. Moreover, the gold particles were synthesized without any additional reducing, stabilizing, or capping agents, thus eliminating the additional step of chemical synthesis, thereby overcoming the limitations. The mechanism behind the synthesis remains to be elucidated. Previous studies have suggested that the cell-free supernatant containing extracellular enzymes and proteins plays a major role in the reduction of HAuCl4·3H2O to gold nanoparticles and in stabilizing the synthesized product (Singh et al. 2015c). The purity of the biosynthesized gold nanoparticles was examined by EDX (Figure 4a). The EDX spectrum displayed an optical absorption band peak at 2.3 keV, which is the characteristic peak of nanosized metallic gold and corresponds to the surface plasmon resonance property of the gold nanoparticles. Due to the use of the carbon-coated copper TEM grid, the carbon and copper signals originated in the low-energy part of the spectrum. Similar results have been observed in the study of biosynthesized gold nanoparticles using ginseng root extract (Singh et al. 2015b). The results of elemental mapping of the biosynthesized F-shaped gold nanoparticles showed the distribution of elements in the electron micrograph of purified gold nanoparticles. Figure 4b corresponds to the electron micrograph of
partially purified gold nanoparticles, and Figure 4c reflects the distribution of elemental gold in the electron micrograph. The results obtained demonstrated that the deposition of gold nanoparticles was 69.92%. The distribution of elemental gold was clearly visible in the elemental maps, and was found to be the predominant element in the respective nanoproduct. Thus, the results of elemental mapping further confirmed the F-shape of the gold nanoparticles. The previous studies have demonstrated the elemental mapping results to characterize the nanoparticle distribution in the relevant nanoproducts. The results obtained here are similar to the previously reported work (Singh et al. 2015a). Figure 5 shows the X-ray
Figure 5. XRD spectrum of F-shaped gold nanoparticles.
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F-shaped gold nanoparticles 5
Figure 6. Particles size distribution of F-shaped gold nanoparticles according to volume (a), intensity (b) and number (c).
diffraction pattern of gold nanoparticles, exhibiting intense peaks in the whole spectrum of 2q value ranging from 20–80, and this pattern is similar to that of the Braggs’s reflection of gold nanocrystals. The characteristic peaks of gold nanocrystals corresponded to (111), (200), (220), and (311). The result indicates the crystalline nature of the F-shaped gold nanoparticles. The XRD results obtained are in the line of previously reported work, which displayed the similar peaks of crystalline gold nanoparticles (Jena and Raj 2007, Singh et al. 2015b). The particle size distribution profile according to volume, intensity, and number of nanoparticles shows that the size of F-shaped gold nanoparticles ranges from 50–150 nm, with an average diameter of 106 nm and a PDI 0.178 (Figure 6a–c). Thus, B. indica DC1 was demonstrated to be suitable for the simple, green, non-hazardous, economical, and eco-friendly synthesis of F-shaped gold nanoparticles without the use of any additional reducing, capping, or stabilizing agent. In the results of the stability test, there was no observable variation in the UV–Vis spectrum obtained after the incubation of reaction mixture at different time intervals, which indicates the stable nature of the F-shaped gold nanoparticles synthesized. In addition, no major change in the wavelength was detected before and after the addition of sodium hydroxide, which further confirmed the stability.
Conclusion The most important advantage of this approach includes the possibility of obtaining F-shaped gold nanoparticles using B. indica DC1. The F-shaped gold nanoparticles were synthesized by an eco-friendly and economical method, eliminating the hazardous effects of physiochemical synthesis. Moreover, the nanoparticles were synthesized without any
extra reducing and capping agents, thus eliminating the extra step in physiochemical synthesis. Furthermore, the methodology can be exploited to scale-up production at a large industrial scale, due to the ease of downstream processing. The biosynthesized F-shaped gold nanoparticles may have the potential for multifunctional use, when compared to spherical nanoparticles, for medical applications such as targeting, diagnosis, photo imaging, and drug delivery. The F-shape has additional significance for optical and electrical applications. Thus, as compared to the conventional methods, a green, time-resolved, and economically viable methodology for the synthesis of F-shaped nanoparticles was developed.
Highlights •• Microbial synthesis of F-shaped gold nanoparticles was achieved. •• The F-shaped gold nanoparticles were characterized by UV–Vis and FE–TEM. •• The nanoparticles were further characterized by EDX, elemental mapping, and XRD. •• The F-shaped gold nanoparticles were 30–100 nm in size. •• The particles were stable and monodisperse.
Acknowledgments This research was supported by the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (KIPET NO: 313038-03-2-SB010) and the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ009529032014), Republic of Korea.
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Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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