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Extracellular synthesis of silver bionanoparticles from Aspergillus clavatus and its antimicrobial activity against MRSA and MRSE
Colloids and Surfaces B: Biointerfaces 77 (2010) 214–218
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Extracellular synthesis of silver bionanoparticles from Aspergillus clavatus and its antimicrobial activity against MRSA and MRSE M. Saravanan a,∗ , Anima Nanda b a b
Department of Biotechnology, SRM University, SRM Nagar, Kattankulathur, Chennai, India Department of Biomedical Engineering, Sathyabama University, Chennai, India
a r t i c l e
i n f o
Article history: Received 13 September 2009 Received in revised form 9 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: Bionanoparticles UV–visible spectroscopy Atomic Force Microscopy Extracellular synthesis
a b s t r a c t New enzymatic approaches using bacteria and fungi for the synthesis of nanoparticles in both intraand extracellular are playing an advanced key role in pharmacotherapeutics. In the present study we have reported on the use of fungus Aspergillus clavatus for the extracellular synthesis of bionanoparticles from silver nitrate (AgNO3 ) solution. The bionanoscale particles were characterized by UV–visible spectroscopy, thin layer chromatography, atomic force microscopy (AFM) and FTIR. The synthesized bionanoscale particle showed a maximum absorption in the visible region of 420 nm. The AFM study of bionanoscale particle ranged in the size of 550–650 nm. The analysis was carried out by TLC and FTIR to identify the biomolecules responsible for the bioreduction of silver ion and capping of the bioreduced silver nanoparticles. The present study analyzes the antimicrobial activity of the silver nanoparticles synthesized from A. clavatus against MRSA and MRSE, which showed the maximum activity against MRSA, followed by MRSE. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nanobiotechnology is an upcoming and fast developing field with potential application especially for human therapeutics. The crux of nanotechnology lies on the novel means of synthesizing nanoscale particles through biological systems for reliable and environmental friendly process [1]. Many organisms including unicellular and multicellular microorganisms have been explored as a potential biofactory for the synthesis of metallic nanoparticles (cadmium sulfide, gold and silver) either intracellularly or extracellularly [2–7]. Recently many studies have been conducted to explore the synthesis of nanoparticles used by microorganisms as a potential, biosources, such as Au and Ag. Fusarium species play a vital role in synthesizing nanoparticles when exposed to Au and Ag ions [8,9]. Holmes et al. in 1995 [10] have shown that the bacteria Klebsiella aerogenes can be used for intracellular synthesis of Cds nanoparticles. Recently the studies done by Nanda and Saravanan in 2009 have proved the antimicrobial activity of silver nanoparticles, synthesized extracellularly from Staphylococcus aureus against MRSA and MRSE. Souza et al. in 2004 [11] showed silver nanoparticles, like its bulk counterpart, are an effective antimicrobial agent against various pathogenic microorganisms. Shrivastava et al. in 2007 [12] have reported the silver nanoparticles in the range of 10–15 nm
∗ Corresponding author. Tel.: +91 09443077097; fax: +91 044 27453903. E-mail address: [email protected] (M. Saravanan). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.01.026
with increased stability and enhanced antimicrobial potency. In the present investigation we have reported the extracellular synthesis, of highly stable bionanoparticles using Aspergillus clavatus and the evaluation of antimicrobial activity against various human pathogenic bacteria, particularly MRSA and MRSE organisms. The study also includes the characterizations of bionanoparticles by UV–visible spectrophotometer, thin layer chromatography (TLC), atomic force microscopy (AFM) and FTIR spectral analysis. 2. Experimental details 2.1. Media and chemicals All the media components and analytical reagents were purchased from Hi-Media Laboratories Pvt. Ltd. (Mumbai, India) and Sigma Chemicals (St. Louis, USA). 2.2. Microorganisms The fungus A. clavatus isolated from animal manure sedimented soil was maintained on potato dextrose agar (PDA) medium at 28◦ C and stored at 4◦ C for further study. The isolated fungus was identified on the basis of their morphological characteristics and microscopic examination. The identified strains were subcultured time to time to regulate viability in the Microbiology Laboratory, Department of Biotechnology, SRM University, Chennai, India during the study period.
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2.3. Extracellular synthesis of Ag bionanoparticles
2.4. Characterization of Ag bionanoparticles
The fungal strains A. clavatus are freshly inoculated on a liquid media containing (g/l) KH2 PO4 , 7.0; K2 HPO4 , 2.0; MgSO4 ·7H2 O, 0.1; (NH4 )2 SO4 , 1.0; yeast extract, 0.6; and glucose, 10.0, in an Erlenmeyer flask. The flaks were incubated on orbital shaker at 25 ◦ C and agitated at 150 rpm for 72 h. The biomass was harvested after 72 h of growth by sieving through a plastic sieves or Whatman No. 1 filter paper, followed by extensive washing with distilled water to remove any medium components from the biomass. Typically 20 g of fresh and clean biomass was taken into Erlenmeyer flask containing 200 ml of milli-Q deionized water (Millipore Water Unit, Bangalore, India) and the flaks were incubated at 25 ◦ C for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through Whatman No. 1 filter paper. 50 ml of cell filtrate was taken into 250 ml of Erlenmeyer flask and mixed with 1 mM AgNO3 (0.017 g AgNO3 /100 ml) as final concentration. The flasks were incubated at 25◦ C in a dark room condition for 120 h. The control was maintained (without the addition of AgNO3 , only cell filtrate) with the experimental flask. The brownish yellow colour solution of Ag bionanoparticles was stored in screw capped vials under ambient condition for future experiments.
The synthesized Ag bionanoparticles were first characterized by Elico UV–visible spectrophotometer in the range of 250–650 nm. (Elico Ltd., Bangalore), using a quartz cuvette with the control as the reference. The surface plasmon resonance peaks were noted around 420–430 nm region. The silver bionanoparticles were kept at room temperature for 3 months to test their stability, and the characterization of the stabilized nanoparticles was carried out using TLC. This was based on the principle that bionanoparticles get separated on basis of differential adsorption on to silica gel. The standard solvent system of N-butanol, acetic acid and water (8:2:2 v/v) was used as a mobile phase. The bionanoparticles were spotted on the TLC plate coated with thin layer of silica gel (G Merck). The sample spotted on the TLC plate was kept inside the chromatographic chamber containing solvent and allowed to develop chromatogram for 20–30 min. The TLC plate removed from the chamber was allowed to dry after 30 min by keeping in hot air oven at 60 ◦ C for 10 min, then the developer (0.3% Ninhydrin) was sprayed and dried at 70 ◦ C for 20 min. The activated TLC plate was observed to elucidate the presence of bionanoparticles.
Fig. 1. Fungal filtrate of Aspergillus clavatus with silver nitrate (a) and control flask (b) after 24 h of reaction with silver nitrate.
Fig. 2. The UV–visible spectrum recorded in the aqueous solution of 1 mM AgNO3 with the fungal filtrate. Curves A, B, C, D and E correspond to readings taken at 24 h, 48 h, 72 h, 96 h and 120 h, respectively. The peak was noted around 420 nm.
Fig. 3. Thin layer chromatographic separation of silver bionanoparticles synthesized by Aspergillus clavatus.
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The morphological characterization of the synthesized bionanoparticles was studied using atomic force Microscopy (Ajilent Technologies) in the contact mode. The sample preparations for the AFM studies were done by dissolving a bionanoparticles samples with acetone and spin coating the sample using apex instruments spin coater at a maximum speed of 9000 rpm. The sample was then dried for 30 min before the studies were conducted. The size and morphology of Ag bionanoparticles was determined. Further characterization of Ag bionanoparticles involved Fourier Transform Infrared Spectroscopy (FTIR) (Perkin-Elmer, Germany) by scanning the spectrum in the range of 450–4000 cm−1 at a resolution of 4 cm−1 2.5. Analysis of antimicrobial activity of Ag bionanoparticles (Ag-BNps) 2.5.1. Well diffusion method The antibacterial activity of the Ag bionanoparticles samples was assayed by following the standard Nathan’s Agar Well Diffusion (NAWD) technique [13]. Four wells of 6 mm diameter were made on the pre-poured Muller Hinton Agar (MHA) plates. These MHA plates were inoculated by swabbing the 18- to 24-h-old test bacterial suspensions to create a confluent lawn of bacterial growth. The Ag bionanoparticles (5 l, 10 l and 15 l) were loaded on to the each of the well. Wells without the extracts were maintained as control. After 20–24 h of incubation at 35 ◦ C room temperature, the susceptibility of the test organisms was determined by measuring the diameter of the zone of inhibition around each well to the nearest mm. 2.5.2. Disc diffusion method The antibacterial activity of the Ag bionanoparticles samples was assayed by following the standard Kirby–Bauer disc diffusion method. Muller Hinton agar was prepared and poured on sterile Petri plates. These MHA plates were inoculated by swabbing the 18to 24-h-old test bacterial suspensions to create a confluent lawn of bacterial growth. Paper disc of 6 mm dimension was impregnated with Ag bionanoparticles in different concentrations (5 l, 10 l, and 15 l). The discs were gently pressed to have a better contact with Muller Hinton Agar. Plates were incubated in inverted position for 18 h at 35 ◦ C and the susceptibility of the test organisms was
determined by measuring the diameter of the zone of inhibition around each disc to the nearest mm. 3. Result and discussion Nanotechnology is a fast emerging discipline in the field of bioscience. A comprehensive study of extracellular synthesis of silver bionanoparticles was carried out in this research work. The fungal biomass after 120 h of incubation was filtered and the filtrate was treated with AgNO3 . The reaction started after 24 h of incubation in dark condition, with change in colour of cell filtrate from pale yellow to brownish yellow, indicating the formation of Ag bionanoparticles (Fig. 1a and b) which correlated with the results obtained by Mukherjee et al. [5]. There was no colour change observed in the control flask incubated in the same environment. Further confirmation of the presence of silver nanoparticles was performed by thin layer chromatography (TLC). The TLC was done on silica gel (G Merck) plates using a mixture of N-butanol, acetic acid and water (8:2:2 v/v) as a solvent and sprayed using Ninhydrin (0.3%) as a developer. The run samples were visualized for the confirmation of the presence of bioactive AgNPs in the reaction mixture (Fig. 3). This corroborates with the results obtained by Gopalakrishnan et al. [14]. Fig. 2 shows the confirmation of formation and stability of AgNPs in the colloidal solution monitored using UV–visible spectral analysis. It was observed that the fungal cell filtrate treated with AgNO3 (1 mM) showed the peak at around 420 nm. This was very specific for silver nanoparticles [15]. FTIR spectroscopy analysis were carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced silver nanoparticles synthesized by fungal cell filtrate. Fig. 4 shows FTIR spectra obtained by Ag-BNps synthesized from A. clavatus where the absorption peaks were located at about 735, 1045, 1645, 2134, and 3400 in the region 450–4000 cm−1 . The FTIR spectral analysis revealed the presence of –C–O–C– and –C C– functional groups, which may be present between amino acid residues and protein synthesized during Ag-BNps. Our results corroborate with Gole et al. [16], they reported that the proteins can bind to nanoparticles through the functional group and it increases the stabilization of nanoparticles. The bond or functional groups such as –C–O–C– and –C C– are derived from the heterocyclic compounds like protein, which are present in the fungal extract and are
Fig. 4. FTIR spectrum recorded from silver bionanoparticles synthesized using Aspergillus clavatus.
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217
Fig. 5. AFM image of silver bionanoparticles on agglomerated silver synthesized by Aspergillus clavatus and corresponding line profile for size determination.
Table 1 Analysis of antimicrobial activity of silver bionanoparticles against MRSA and MRSE. S. No.
Pathogenic bacteria
Concentration of AgNPs used
Mean zone of inhibition (mm) in well and disc diffusion methods
1
MRSA (methicillin resistant Staphylococcus aureus)
Control 5 l 10 l 15 l
00 00 13 20.5
2
MRSE (methicillin resistant Staphylococcus epidermidis)
Control 5 l 10 l 15 l
00 00 5 19
Fig. 6. Antimicrobial activity of silver bionanoparticles against MRSA and MRSE tested by well diffusion method.
capping ligands of the nanoparticles, was reported by Sastry et al. [9] and Sanghi and Verma [17]. The topography and morphology of Ag-BNps were studied using atomic force microscopy in the contact mode. It was noticed that irregular Ag-BNps on agglomerated silver as shown in Fig. 5. The particle size was determined by the line profile. The Ag-BNps were measured and found in the range of 550–650 nm in diameter. The efficacy of synthesized nanoparticles was tested against MRSA and MRSE by disc and well diffusion methods (Figs. 6 and 7). Different concentration levels were tested in the well, as well as in disc to confirm the zone of inhibition. The maximum antimicrobial activity was recorded in MRSA followed by MRSE. The mean zone of inhibition in disc and well diffusion methods were recorded for MRSA and MRSE (Table 1). The efficacy of bionanoparticles tested against MRSA at 10 l concentration revealed the zone of inhibition of 13 mm in diameter, whereas 15 l concentration of
bionanoparticles revealed the zone of inhibition of 20.5 mm in diameter. In the case of MRSE 10 l concentration of silver bionanoparticles revealed the zone of inhibition of 5 mm diameter, whereas 15 l concentration produced the zone of inhibition of 19 mm in diameter. In the case of 5 l concentration, there was no zone of inhibition for both MRSA and MRSE. This corroborates with the results obtained by Nanda and Saravanan [18] which proved the antimicrobial activity of silver bionanoparticles against MRSA and MRSE synthesized form S. aureus. The bactericidal effect of silver nanoparticles against multi drug resistant bacteria was confirmed with the earlier work done by Lara et al. [19] and Song et al. [20]. The inhibition zone formed in the screening test indicated that the synthesized AgNPs have antibacterial activity against human pathogenic bacteria (MRSA and MRSE). This shows that the synthesized silver nanostructure by this process is ready for the application in the field of nanomedicine. However, further studies are required on understanding the cellular and molecular mechanism of bionanoparticles synthesis and its antimicrobial activity. Acknowledgements The authors gratefully acknowledge the Management, SRM University (Kattankulathur, Chennai, India) for providing the facilities to perform the research work in the Department of Biotechnology and Nanotechnology Research Centre. The authors would like to acknowledge SAIF (Sophisticated Analytical Instrument Facility) IIT, Chennai, for the FTIR analysis. References
Fig. 7. Antimicrobial activity of silver bionanoparticles against MRSA and MRSE tested by disc diffusion method.
[1] M. Deendayal, E.M. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biotechnol. 69 (2006) 485–492.
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[2] T. Klaus, R. Joerger, E. Olsson, C.G. Granqvist, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 13611. [3] B. Nair, T. Pradeep, Cryst. Growth Des. 2 (2002) 293. [4] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, et al., Nanotechnology 14 (2003) 95. [5] P. Mukherjee, A. Ahmad, D. Mandal, S. Senapati, S.R. Sainkar, M.I. Khan, et al., Nano Lett. 1 (2001) 515; A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar, et al., Colloids Surf. B: Biointerfaces 28 (2003) 313–318. [6] A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, R. Ramani, V. Srinivas, et al., Nanotechnology 14 (2003) 824–828. [7] A. Vigneshwaran, A.A. Kathe, P.V. Varadarajan, R.P. Nachne, R.H. Balasubramanya, Colloids Surf. B: Biointerfaces 53 (2006) 55–59. [8] S. Basavaraja, S.D. Balaji, A. Lagashetty, A.H. Rajasab, A. Venkataraman, Mater. Res. Bull. 43 (2008) 1164–1170. [9] M. Sastry, A. Ahmad, M.I. Khan, R. Kumar, Curr. Sci. 85 (2003) 162–170. [10] J.D. Holmes, P.R. Smith, R. Evans-Gowing, D.J. Richardson, D.A. Russel, J.R. Sodeau, Arch. Microbiol. 163 (1995) 143–147.
[11] G.I.H. Souza, P.D. Marcato, N. Durán, E. Esposito, IX National Meeting of Environmental Microbiology, Curtiba, PR, Brazil, 2004, p. 25 (abstract). [12] S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, D. Dash, Nanotechnology 18 (2007) 225103 (9 pp). [13] P. Nathan, E.J. Law, D.F. Murphy, Burns 4 (1978) 177–178. [14] G. Gopalakrishnan, S. Ramaswamy, U. Memoriya, J. Scan. Probe Microsc. 2 (2007) 58–62. [15] A. Ingle, A. Gade, S. Pierrat, C. Sonnichsen, M. Rai, Curr. Nanosci. 4 (2008) 141–144. [16] A. Gole, C. Dash, V. Ramakrishnan, S.R. Sainkar, A.B. Mandale, M. Rao, M. Sastry, Langmuir 7 (2001) 1674–1679. [17] R. Sanghi, P. Verma, Bioresource Technol. 100 (2009) 501–504. [18] A. Nanda, M. Saravanan, Nanomed. Nanotechol. Biol. Med. 5 (2009) 452–456. ˜ [19] H.H. Lara, N.V. Ayala-Nu˘ınez, L.C.I. Turrent, C. R. Padilla, World J. Microbiol. Biotechnol. (2009) in press. [20] H.Y. Song, K.K. Ko, I.H. Oh, B.T. Lee, Eur. Cell Mater. 11 (2006) 58.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Extracellular synthesis of silver bionanoparticles from Aspergillus clavatus and its antimicrobial activity against MRSA and MRSE M. Saravanan a,∗ , Anima Nanda b a b
Department of Biotechnology, SRM University, SRM Nagar, Kattankulathur, Chennai, India Department of Biomedical Engineering, Sathyabama University, Chennai, India
a r t i c l e
i n f o
Article history: Received 13 September 2009 Received in revised form 9 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: Bionanoparticles UV–visible spectroscopy Atomic Force Microscopy Extracellular synthesis
a b s t r a c t New enzymatic approaches using bacteria and fungi for the synthesis of nanoparticles in both intraand extracellular are playing an advanced key role in pharmacotherapeutics. In the present study we have reported on the use of fungus Aspergillus clavatus for the extracellular synthesis of bionanoparticles from silver nitrate (AgNO3 ) solution. The bionanoscale particles were characterized by UV–visible spectroscopy, thin layer chromatography, atomic force microscopy (AFM) and FTIR. The synthesized bionanoscale particle showed a maximum absorption in the visible region of 420 nm. The AFM study of bionanoscale particle ranged in the size of 550–650 nm. The analysis was carried out by TLC and FTIR to identify the biomolecules responsible for the bioreduction of silver ion and capping of the bioreduced silver nanoparticles. The present study analyzes the antimicrobial activity of the silver nanoparticles synthesized from A. clavatus against MRSA and MRSE, which showed the maximum activity against MRSA, followed by MRSE. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nanobiotechnology is an upcoming and fast developing field with potential application especially for human therapeutics. The crux of nanotechnology lies on the novel means of synthesizing nanoscale particles through biological systems for reliable and environmental friendly process [1]. Many organisms including unicellular and multicellular microorganisms have been explored as a potential biofactory for the synthesis of metallic nanoparticles (cadmium sulfide, gold and silver) either intracellularly or extracellularly [2–7]. Recently many studies have been conducted to explore the synthesis of nanoparticles used by microorganisms as a potential, biosources, such as Au and Ag. Fusarium species play a vital role in synthesizing nanoparticles when exposed to Au and Ag ions [8,9]. Holmes et al. in 1995 [10] have shown that the bacteria Klebsiella aerogenes can be used for intracellular synthesis of Cds nanoparticles. Recently the studies done by Nanda and Saravanan in 2009 have proved the antimicrobial activity of silver nanoparticles, synthesized extracellularly from Staphylococcus aureus against MRSA and MRSE. Souza et al. in 2004 [11] showed silver nanoparticles, like its bulk counterpart, are an effective antimicrobial agent against various pathogenic microorganisms. Shrivastava et al. in 2007 [12] have reported the silver nanoparticles in the range of 10–15 nm
∗ Corresponding author. Tel.: +91 09443077097; fax: +91 044 27453903. E-mail address: [email protected] (M. Saravanan). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.01.026
with increased stability and enhanced antimicrobial potency. In the present investigation we have reported the extracellular synthesis, of highly stable bionanoparticles using Aspergillus clavatus and the evaluation of antimicrobial activity against various human pathogenic bacteria, particularly MRSA and MRSE organisms. The study also includes the characterizations of bionanoparticles by UV–visible spectrophotometer, thin layer chromatography (TLC), atomic force microscopy (AFM) and FTIR spectral analysis. 2. Experimental details 2.1. Media and chemicals All the media components and analytical reagents were purchased from Hi-Media Laboratories Pvt. Ltd. (Mumbai, India) and Sigma Chemicals (St. Louis, USA). 2.2. Microorganisms The fungus A. clavatus isolated from animal manure sedimented soil was maintained on potato dextrose agar (PDA) medium at 28◦ C and stored at 4◦ C for further study. The isolated fungus was identified on the basis of their morphological characteristics and microscopic examination. The identified strains were subcultured time to time to regulate viability in the Microbiology Laboratory, Department of Biotechnology, SRM University, Chennai, India during the study period.
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2.3. Extracellular synthesis of Ag bionanoparticles
2.4. Characterization of Ag bionanoparticles
The fungal strains A. clavatus are freshly inoculated on a liquid media containing (g/l) KH2 PO4 , 7.0; K2 HPO4 , 2.0; MgSO4 ·7H2 O, 0.1; (NH4 )2 SO4 , 1.0; yeast extract, 0.6; and glucose, 10.0, in an Erlenmeyer flask. The flaks were incubated on orbital shaker at 25 ◦ C and agitated at 150 rpm for 72 h. The biomass was harvested after 72 h of growth by sieving through a plastic sieves or Whatman No. 1 filter paper, followed by extensive washing with distilled water to remove any medium components from the biomass. Typically 20 g of fresh and clean biomass was taken into Erlenmeyer flask containing 200 ml of milli-Q deionized water (Millipore Water Unit, Bangalore, India) and the flaks were incubated at 25 ◦ C for 72 h and agitated in the same condition as described earlier. After incubation, the cell filtrates were obtained by passing it through Whatman No. 1 filter paper. 50 ml of cell filtrate was taken into 250 ml of Erlenmeyer flask and mixed with 1 mM AgNO3 (0.017 g AgNO3 /100 ml) as final concentration. The flasks were incubated at 25◦ C in a dark room condition for 120 h. The control was maintained (without the addition of AgNO3 , only cell filtrate) with the experimental flask. The brownish yellow colour solution of Ag bionanoparticles was stored in screw capped vials under ambient condition for future experiments.
The synthesized Ag bionanoparticles were first characterized by Elico UV–visible spectrophotometer in the range of 250–650 nm. (Elico Ltd., Bangalore), using a quartz cuvette with the control as the reference. The surface plasmon resonance peaks were noted around 420–430 nm region. The silver bionanoparticles were kept at room temperature for 3 months to test their stability, and the characterization of the stabilized nanoparticles was carried out using TLC. This was based on the principle that bionanoparticles get separated on basis of differential adsorption on to silica gel. The standard solvent system of N-butanol, acetic acid and water (8:2:2 v/v) was used as a mobile phase. The bionanoparticles were spotted on the TLC plate coated with thin layer of silica gel (G Merck). The sample spotted on the TLC plate was kept inside the chromatographic chamber containing solvent and allowed to develop chromatogram for 20–30 min. The TLC plate removed from the chamber was allowed to dry after 30 min by keeping in hot air oven at 60 ◦ C for 10 min, then the developer (0.3% Ninhydrin) was sprayed and dried at 70 ◦ C for 20 min. The activated TLC plate was observed to elucidate the presence of bionanoparticles.
Fig. 1. Fungal filtrate of Aspergillus clavatus with silver nitrate (a) and control flask (b) after 24 h of reaction with silver nitrate.
Fig. 2. The UV–visible spectrum recorded in the aqueous solution of 1 mM AgNO3 with the fungal filtrate. Curves A, B, C, D and E correspond to readings taken at 24 h, 48 h, 72 h, 96 h and 120 h, respectively. The peak was noted around 420 nm.
Fig. 3. Thin layer chromatographic separation of silver bionanoparticles synthesized by Aspergillus clavatus.
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The morphological characterization of the synthesized bionanoparticles was studied using atomic force Microscopy (Ajilent Technologies) in the contact mode. The sample preparations for the AFM studies were done by dissolving a bionanoparticles samples with acetone and spin coating the sample using apex instruments spin coater at a maximum speed of 9000 rpm. The sample was then dried for 30 min before the studies were conducted. The size and morphology of Ag bionanoparticles was determined. Further characterization of Ag bionanoparticles involved Fourier Transform Infrared Spectroscopy (FTIR) (Perkin-Elmer, Germany) by scanning the spectrum in the range of 450–4000 cm−1 at a resolution of 4 cm−1 2.5. Analysis of antimicrobial activity of Ag bionanoparticles (Ag-BNps) 2.5.1. Well diffusion method The antibacterial activity of the Ag bionanoparticles samples was assayed by following the standard Nathan’s Agar Well Diffusion (NAWD) technique [13]. Four wells of 6 mm diameter were made on the pre-poured Muller Hinton Agar (MHA) plates. These MHA plates were inoculated by swabbing the 18- to 24-h-old test bacterial suspensions to create a confluent lawn of bacterial growth. The Ag bionanoparticles (5 l, 10 l and 15 l) were loaded on to the each of the well. Wells without the extracts were maintained as control. After 20–24 h of incubation at 35 ◦ C room temperature, the susceptibility of the test organisms was determined by measuring the diameter of the zone of inhibition around each well to the nearest mm. 2.5.2. Disc diffusion method The antibacterial activity of the Ag bionanoparticles samples was assayed by following the standard Kirby–Bauer disc diffusion method. Muller Hinton agar was prepared and poured on sterile Petri plates. These MHA plates were inoculated by swabbing the 18to 24-h-old test bacterial suspensions to create a confluent lawn of bacterial growth. Paper disc of 6 mm dimension was impregnated with Ag bionanoparticles in different concentrations (5 l, 10 l, and 15 l). The discs were gently pressed to have a better contact with Muller Hinton Agar. Plates were incubated in inverted position for 18 h at 35 ◦ C and the susceptibility of the test organisms was
determined by measuring the diameter of the zone of inhibition around each disc to the nearest mm. 3. Result and discussion Nanotechnology is a fast emerging discipline in the field of bioscience. A comprehensive study of extracellular synthesis of silver bionanoparticles was carried out in this research work. The fungal biomass after 120 h of incubation was filtered and the filtrate was treated with AgNO3 . The reaction started after 24 h of incubation in dark condition, with change in colour of cell filtrate from pale yellow to brownish yellow, indicating the formation of Ag bionanoparticles (Fig. 1a and b) which correlated with the results obtained by Mukherjee et al. [5]. There was no colour change observed in the control flask incubated in the same environment. Further confirmation of the presence of silver nanoparticles was performed by thin layer chromatography (TLC). The TLC was done on silica gel (G Merck) plates using a mixture of N-butanol, acetic acid and water (8:2:2 v/v) as a solvent and sprayed using Ninhydrin (0.3%) as a developer. The run samples were visualized for the confirmation of the presence of bioactive AgNPs in the reaction mixture (Fig. 3). This corroborates with the results obtained by Gopalakrishnan et al. [14]. Fig. 2 shows the confirmation of formation and stability of AgNPs in the colloidal solution monitored using UV–visible spectral analysis. It was observed that the fungal cell filtrate treated with AgNO3 (1 mM) showed the peak at around 420 nm. This was very specific for silver nanoparticles [15]. FTIR spectroscopy analysis were carried out to identify the biomolecules responsible for the reduction of Ag+ ions and capping of the bioreduced silver nanoparticles synthesized by fungal cell filtrate. Fig. 4 shows FTIR spectra obtained by Ag-BNps synthesized from A. clavatus where the absorption peaks were located at about 735, 1045, 1645, 2134, and 3400 in the region 450–4000 cm−1 . The FTIR spectral analysis revealed the presence of –C–O–C– and –C C– functional groups, which may be present between amino acid residues and protein synthesized during Ag-BNps. Our results corroborate with Gole et al. [16], they reported that the proteins can bind to nanoparticles through the functional group and it increases the stabilization of nanoparticles. The bond or functional groups such as –C–O–C– and –C C– are derived from the heterocyclic compounds like protein, which are present in the fungal extract and are
Fig. 4. FTIR spectrum recorded from silver bionanoparticles synthesized using Aspergillus clavatus.
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Fig. 5. AFM image of silver bionanoparticles on agglomerated silver synthesized by Aspergillus clavatus and corresponding line profile for size determination.
Table 1 Analysis of antimicrobial activity of silver bionanoparticles against MRSA and MRSE. S. No.
Pathogenic bacteria
Concentration of AgNPs used
Mean zone of inhibition (mm) in well and disc diffusion methods
1
MRSA (methicillin resistant Staphylococcus aureus)
Control 5 l 10 l 15 l
00 00 13 20.5
2
MRSE (methicillin resistant Staphylococcus epidermidis)
Control 5 l 10 l 15 l
00 00 5 19
Fig. 6. Antimicrobial activity of silver bionanoparticles against MRSA and MRSE tested by well diffusion method.
capping ligands of the nanoparticles, was reported by Sastry et al. [9] and Sanghi and Verma [17]. The topography and morphology of Ag-BNps were studied using atomic force microscopy in the contact mode. It was noticed that irregular Ag-BNps on agglomerated silver as shown in Fig. 5. The particle size was determined by the line profile. The Ag-BNps were measured and found in the range of 550–650 nm in diameter. The efficacy of synthesized nanoparticles was tested against MRSA and MRSE by disc and well diffusion methods (Figs. 6 and 7). Different concentration levels were tested in the well, as well as in disc to confirm the zone of inhibition. The maximum antimicrobial activity was recorded in MRSA followed by MRSE. The mean zone of inhibition in disc and well diffusion methods were recorded for MRSA and MRSE (Table 1). The efficacy of bionanoparticles tested against MRSA at 10 l concentration revealed the zone of inhibition of 13 mm in diameter, whereas 15 l concentration of
bionanoparticles revealed the zone of inhibition of 20.5 mm in diameter. In the case of MRSE 10 l concentration of silver bionanoparticles revealed the zone of inhibition of 5 mm diameter, whereas 15 l concentration produced the zone of inhibition of 19 mm in diameter. In the case of 5 l concentration, there was no zone of inhibition for both MRSA and MRSE. This corroborates with the results obtained by Nanda and Saravanan [18] which proved the antimicrobial activity of silver bionanoparticles against MRSA and MRSE synthesized form S. aureus. The bactericidal effect of silver nanoparticles against multi drug resistant bacteria was confirmed with the earlier work done by Lara et al. [19] and Song et al. [20]. The inhibition zone formed in the screening test indicated that the synthesized AgNPs have antibacterial activity against human pathogenic bacteria (MRSA and MRSE). This shows that the synthesized silver nanostructure by this process is ready for the application in the field of nanomedicine. However, further studies are required on understanding the cellular and molecular mechanism of bionanoparticles synthesis and its antimicrobial activity. Acknowledgements The authors gratefully acknowledge the Management, SRM University (Kattankulathur, Chennai, India) for providing the facilities to perform the research work in the Department of Biotechnology and Nanotechnology Research Centre. The authors would like to acknowledge SAIF (Sophisticated Analytical Instrument Facility) IIT, Chennai, for the FTIR analysis. References
Fig. 7. Antimicrobial activity of silver bionanoparticles against MRSA and MRSE tested by disc diffusion method.
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