pubs.acs.org/Langmuir © 2009 American Chemical Society
Intracellular Biogenic Silver Nanoparticles for the Generation of Carbon Supported Antiviral and Sustained Bactericidal Agents P. S. Vijayakumar and B. L. V. Prasad* Materials Chemistry Division, National Chemical Laboratory, Pune - 08, India Received March 24, 2009. Revised Manuscript Received July 30, 2009 Intracellular silver nanoparticles produced by exposing silver ions to the fungus Aspergillus ochraceus were heattreated in nitrogen environment to yield silver nanoparticles embedded in carbonaceous supports. This carbonaceous matrix embedded silver nanoparticles showed antimicrobial properties against both bacteria (Gram-positive and Gramnegative) and virus (M 13 phage virus). The bactericidal effects were noticed even after washing and repeated exposure of these carbon supported silver nanoparticles to fresh bacterial cultures, revealing their sustained activity.
Introduction The antibacterial properties of silver nanoparticles (AgNPs) have been the subject of many studies.1 They have gained widespread attention as nanomolar concentrations of AgNPs were found to be as effective as micromolar concentrations of silver ions.2 Moreover, silver ions were susceptible to losses through washing, and hence, the focus has shifted to immobilized AgNPs that could show sustained biocidal activity.1e,3 For example, polymers modified with lipids were shown to generate in situ nanoparticles through lipid autoxidation, and such in situ generated solutions were used to coat substrates to reveal scratch-proof antimicrobial coatings.4 Another example that has relevance in the area of biomedical applications was silver carbonate nanoparticles immobilized on alumina nanoneedles having antibacterial activity proportional to the solubility5 and gold nanoparticles stabilized with the dendrons and cationic amphiphiles that show better interaction with biological and artificial membranes.6 On the other hand, carbon/activated carbon has been generally considered better for immobilization of AgNPs due to the possibility of increased adsorption of various organics apart from removing bacteria that makes them popular choices for water purification applications.7 However, the AgNPs have to be immobilized strongly on the carbon support, as improper attachment to the matrix runs the risk of the nanomaterial getting *E-mail:
[email protected]. Phone 91-20-25902013 and Fax 91-2025902636. (1) (a) Smetana, A. B.; Klabunde, K. J.; Marchin, G. R.; Sorensen, C. M. Langmuir 2008, 24, 7457–7464. (b) Ramstedt, M.; Cheng, N.; Azzaroni, O.; Mossialos, D.; Mathieu, H. J.; Huck, W. T. S. Langmuir 2007, 23, 3314–3321. (c) Dias, H. V. R.; Batdorf, K. H.; Fianchini, M.; Diyabalanage, H. V. K.; Carnahan, S.; Mulcahy, R.; Rabiee, A.; Nelson, K.; Waasbergen, L. G. J. Inorg. Biochem. 2006, 100, 158–160. (d) Sambhy, V.; MacBride, M. M.; Peterson, B. R.; Sen, A. J. Am. Chem. Soc. 2006, 128, 9798–9808. (e) Shi, Z.; Neoh, K. G.; Kang, E. T. Langmuir 2004, 20, 6847–6852. (f) Balogh, L.; Swanson, D. R.; Tomalia, D. A.; Hagnauer, G. L.; McManus, A. T. Nano Lett. 2001, 1, 18–21. (2) Lok, C. N.; Ho, C. M.; Chen, R.; He, Q. Y.; Yu, W. Y.; Sun, H.; Tam, P. K. H.; Chiu, J. F.; Che, C. M. J. Proteome Res. 2006, 5, 916–924. (3) Melaiye, A.; Sun, Z.; Hindi, K.; Milsted, A.; Ely, D.; Reneker, D. H.; Tessier, C. A.; Youngs, W. J. J. Am. Chem. Soc. 2005, 127, 2285–2291. (4) Kumar, A.; Vemula, P. K.; Ajayan, P. M.; John, G. Nat. Mater. 2008, 7, 236– 241. (5) Buckley, J. J.; Gai, P. L.; Lee, A. F.; Olivi, L.; Wilson, K. Chem. Commun. 2008, 4013–4015. (6) (a) Leroueil, P. R.; Berry, S. A.; Duthie, K.; Han, G.; Rotello, V. M.; McNerny, D. Q.; Baker, J. R.; Orr, B. G., Jr.; Holl, M. M. B Nano Lett. 2008, 8, 420–424. (b) Bhattacharya, S.; Srivastava, A. Langmuir 2003, 19, 4439–4447. (7) (a) Shi, Z.; Neoh, K. G.; Kang, E. T. Ind. Eng. Chem. Res. 2007, 46, 439–445. (b) Quinlivan, P. A.; Li, L.; Knappe, D. R. Water Res. 2005, 39, 1663–1673.
Langmuir 2009, 25(19), 11741–11747
washed away when exposed to solvent media as observed by Fu and co-workers.8 Recently, carbon-supported AgNPs were prepared by impregnating Agþ ions on carbon fibers and then reducing Agþ ions in the composite to metallic form. While these composite materials showed excellent antibacterial properties, only ∼30% of the silver used in the synthesis protocol was immobilized in the carbon, as the rest got washed away.9 Consequently, there is a strong requirement for new methods of carbon immobilization that not only provide strong support to the nanoparticles but display bactericidal property in a sustainable way. We envisaged biogenic intracellular nanoparticles obtained from a microbial route as one such source.10 Intracellular synthesis of nanoparticles was first reported by Beveridge and co-workers11 and Klaus et al.12 in bacteria. While intracellular synthesis of nanoparticles was clearly established by Klaus and co-workers,12 the mechanism of synthesis is still being investigated with some recent studies suggesting the role of silver resistant gene for the reduction of Agþ ions to AgNPs.13 Subsequent to the initial studies on bacteria,10-15 screening and documentation of several eukaryotic fungal genera especially Verticellium sp. and actinomycetes for intracellular nanoparticles synthesis were done extensively.16 The extracellular synthesis of nanoparticles with Fusarium oxysporum and Trichothecium sp. followed these studies by challenging these fungi with various combincations of metal ions to produce different metal and metal oxide nanoparticles.14,17 (8) Zhang, S.; Fu, R.; Wu, D.; Xu, W.; Ye, Q.; Chen, Z. Carbon 2004, 42, 3209– 3216. (9) Bandyopadhyaya, R.; Sivaiah, M. V.; Shankar, P. A. J. Chem. Technol. Biotechnol. 2008, 83, 1177–1180. (10) Joerger, R.; Klaus, T.; Granqvist, C. G. Adv. Mater. 2000, 12, 407–409. (11) Southam, G.; Beveridge, T. J. Geochim. Cosmochim. Acta 1996, 60, 4369– 4376. (12) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13611–13614. (13) Parikh, R. Y.; Singh, S.; Prasad, B. L. V.; Patole, M. S.; Sastry, M.; Shouche, Y. S. ChemBioChem 2008, 9, 1415–1422. (14) (a) Duran, N.; Marcato, P. D.; Alves, O. L.; De Souza, G. I. H.; Esposito, E. J Nanobiotechnology 2005, 3, 8. (b) Bansal, V.; Rautaray, D.; Ahmad, A.; Sastry, M. J. Mater. Chem. 2004, 14, 3303–3333. (c) Ahmad, A.; Mukherjee, P.; Senapati, S.; Mandal, D.; Khan, M. I.; Kumar, R.; Sastry, M. Colloids Surf., B 2003, 28, 313–318. (15) Nair, B.; Pradeep, T. Cryst. Growth Des. 2002, 2, 293–298. (16) (a) Sastry, M.; Ahmad, A.; Khan, M. I.; Kumar, R. Curr. Sci. 2003, 85, 162– 170. (b) Ahmad, A.; Senapati, S.; Khan, M I.; Kumar, R.; Ramani, R.; Srinivas, V.; Sastry, M. Nanotechnology 2003, 14, 824–828. (c) Chen, J. C.; Lin, Z. H.; Ma, X. X. Lett. Appl. Microbiol. 2003, 37, 105–108. (d) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parishcha, R.; Ajaykumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Nano Lett. 2001, 1, 515–519. (17) Ahmad, A.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. J. Biomed. Nanotechnol. 2005, 1, 47–53.
Published on Web 09/11/2009
DOI: 10.1021/la901024p
11741
Article
Vijayakumar and Prasad
Further, the alloy and shape-controlled nanoparticle synthesis have also been demonstrated.18 In this context, the extracellular synthesis masked further research on intracellular nanoparticles, as finding an application for the intracellular synthesis where nanoparticles were entangled in cellular matrix looked very difficult, though in general more organisms produce intracellular nanoparticulate systems. We envisaged that, after an intracellular synthesis, simple heat treatment under inert conditions would convert biomatrix into a carbonaceous support with embedded nanoparticles. Indeed, not only did we find that such methods could produce nanoparticles immobilized in carbonaceous matrix, but also better immobilization of the intracellulary produced nanoparticles was seen. The porous carbonaceous matrix, on the other hand, enabled better accessibility and sustainable release of the effective ingredients. Generally, procedures such as the arc discharge method were employed to obtain nanoparticles embedded in carbonaceous matrix,19 which are highly energy intensive. In contrast, the present synthetic procedure is relatively simpler and environmentally benign. Two hypotheses were put forward for the bactericidal activity of silver: one is the ability of silver to cause pore formation in cell membrane through the formation of reactive oxygen species (ROS) in the vicinity of bacterial cell membrane and thus increase cell permeability and cell death.20 The second is its interaction with DNA and various cellular enzymes such as cytochrome oxidase, NADH-succinate-dehydrogenase, and DNA that affect the cell division process again leading to cell death.21 Both mechanisms depend on Agþ release. Hence, the nanoparticle prone to aggregation in a high electrolyte condition loses antibacterial activity,22 where again supported materials are believed to fare better. In the present report, the antibacterial properties of the carbon-supported AgNPs were tested against E. coli and Bacillus subtilis cultures (Gram-negative and Gram-positive bacteria, respectively), and as expected, these reveal excellent bactericidal properties for repeated exposures. An interesting addition to our result was the possibility of the supporting carbonaceous matrix acting as bait for the microbes to get attracted to the material and thus exposed to AgNPs.23 Recently, AgNPs also gained importance as antiviral agents.24 Hence, our supported silver particle activity against viruses was also evaluated with plaque assay. Here again, the particles synthesized by us showed good antiviral activity. Presented below are the details of the investigations.
Experimental Section Materials. Maltose, glucose, yeast extract, and peptone were procured from Himedia chemicals. AgNO3 was procured from SRL chemicals. NaBH4 was procured form Sigma Aldrich. Tetracycline hydrochloride was purchased from Himedia. Valcon (18) Senapati, S.; Ahmad, A.; Khan, M. I.; Sastry, M.; Kumar, R. Small 2005, 1, 517–520. (19) (a) Si, P. Z.; Zhang, Z. D.; Geng, D. Y.; You, C. Y.; Zhao, X. G.; Zhang, W. S. Carbon 2003, 41, 247–251. (b) Host, J. J.; Block, J. A.; Parvin, K.; Dravid, V. P. J. Appl. Phys. 1998, 83, 793–801. (20) (a) Gogoi, S. K.; Gopinath, P.; Paul, A.; Ramesh, A.; Ghosh, S. S.; Chattopadhyay, A. Langmuir 2006, 22, 9322–9328. (b) Sondi, I.; S. Sondi, B. S. J. Colloid Interface Sci. 2004, 275, 177–182. (c) Maness, P. C.; Smolinski, S.; Blake, D. M.; Huang, Z.; Wolfrum, E. J.; Jacoby, W. A. Appl. Environ. Microbiol. 1999, 65, 4094–4098. (21) (a) Kumar, R.; Howdle, S.; Munstedt, H. J. Biomed. Mater. Res. 2005, 75B, 311–319. (b) Klueh, U.; Wagner, V.; Kelly, S.; Johnson, A.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 53, 621–631. (22) Lok, C. N.; Ho, C. M.; Chen, R.; He, Q. Y.; Yu, W. Y.; Sun, H.; Tam, P. K. H.; Chiu, J. F.; Che, C. M. J. Biol. Inorg. Chem. 2007, 12, 527–534. (23) Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M. Langmuir 2007, 23, 8670–8673. (24) (a) Rogers, J. V.; Parkinson, C. V.; Choi, Y. W.; Speshock, J. L.; Hussain, S. M. Nanoscale Res. Lett. 2008, 3, 129–133. (b) Sun, R. W. Y.; Chen, R.; Chung, N. P. Y.; Ho, C. M.; Lin, C. L. S.; Che, C. M. Chem. Commun. 2005, 5059–5061.
11742 DOI: 10.1021/la901024p
XC 72 with a specific surface area of 250 m2/g was procured from Cabot Corporation, Boston, Massachusetts. Glutraldehyde was purchased from SD Fine chemicals, Mumbai. Deionized Milli-Q water was used wherever necessary. The bacterial cultures were provided by NCIM (National Centre for Industrial Microorganisms), NCL, India. M13 mp 18 RF DNA was purchased from TAKARA Bio Inc. Instrumental Details. The X-ray diffractograms (XRD) were recorded on a PANanalytical Xpert pro machine using a Cu KR source at operating conditions of 40 mA and 30 kV at a scan rate of 4°/min. TEM samples of carbon-supported AgNPs were prepared by placing drops of their aqueous dispersions over amorphous carbon-coated copper grids and allowing the solvent to air-dry. Transmission electron microscopy (TEM) images were recorded using a Technai G2 F-30 model operated at an accelerating voltage of 300 kV and JEM 2100 instrument operated at 200 kV. Raman spectra were measured in the backscattering configuration using a 514.5 nm Ar laser excitation. The scattered light was analyzed in a Jobin-Yvon HR460 single-grating spectrometer equipped with a charge-coupled array detector and a holographic notch filter (Kaiser Optical Systems, Inc., Ann Arbor, MI). To avoid laser damage to the sample, the experiments were conducted at low laser powers (2 W/cm2). Scanning electron microscopic (SEM) images were recorded from Leica Stereoscan model 440. The silver concentrations were measured with a Chemito atomic absorption spectrometer (AAS) 201 with a silver, hollow cathode lamp. Elemental analysis for C, H, and N was done by EA1108 Elemental Analyzer (Carlo Erba Instruments). Synthesis and Characterization. The fungus that was isolated from a foundry polluted area was identified and registered at Indian type Culture Collection (ITCC) Aspergillus ochraceus 6436. This fungus is already known to grow in metal ions rich concentration.23 In a typical reaction, the isolated fungal spores were inoculated in 500 mL Erlenmeyer flask containing 200 mL of MGYP broth. The spores were allowed to germinate and produce hyphal biomass for three days in a shaker (200 rpm) at 37 °C. It produced 60 g biomass on a wet weight basis that was harvested, and three rounds of washing (1000 rpm, 15 min) were done with autoclaved Milli-Q water under sterile condition. The biomass was then resuspended in 200 mL of 10-3 M AgNO3, followed by incubation in a shaker (200 rpm) for two days at 37 °C. The product biomass was washed with Milli-Q water and heat-treated (600 °C for 6 h) in a tubular furnace under nitrogen flow. It gave 400 mg product that was ground finely and characterized. The silver concentration in this sample was analyzed using AAS and found to be ∼4 wt %. This is denoted as sample 2. For the control experiment, the pure biomass (60 g) without exposure to AgNO3 was collected in the same fashion as described above, and this was again heat-treated, keeping all the conditions same as above. This is denoted as sample 1. Four more control samples were prepared. The first of such controls were prepared by immobilizing AgNO3 on pre-heat-treated biomass. This was done as follows: First, 60 g of wet biomass was collected and was subjected to heat treatment as described in the preparation of sample 1. This carbon material was stirred with 10-3 M AgNO3. More AgNO3 was added at regular intervals, and the intermittent carbon samples were subjected to AAS analysis. The reaction was continued until the amount of silver ions immobilized as analyzed by AAS turned out to be ∼4 wt %. This sample is referred to as sample 3 further in the text. To prepare the next control sample, citrate reduced AgNPs (200 mL 10-4 AgNO3 and 0.01% tri sodium citrate heated for 5 min followed by immediate cooling) were prepared first. These were stirred with heat-treated biomass. More citrate reduced AgNPs were added intermittently and UV-vis spectra of the supernatant were recorded at regular intervals. The reaction (stirring of citrate reduces AgNPs with heat-treated biomass) was stopped when there was no decrease in the intensity of supernatant absorbance peak at 420 nm, which denoted saturation of loading of heat-treated biomass with AgNPs. This is referred to as sample 4. The third control was prepared by Langmuir 2009, 25(19), 11741–11747
Vijayakumar and Prasad Table 1. Sample Identity Number and the Methodology Adopted identity number sample 1 sample 2
sample 3 sample 4 sample 5 sample 6
methodology adopted stepwise Step 1 Wet biomass synthesis Step 2 Heat treatment of the wet biomass Step 1 Wet biomass synthesis Step 2 Incubation of wet biomass in AgNO3 precursor for the intracellular silver nanoparticle synthesis Step 3 Heat treatment of biomass containing insitu intracellularly synthesized AgNPs Step 1 & Step 2 Similar to sample 1 preparation Step 3 AgNO3 incubation with heat treated biomass until the silver ion loading reaches ∼4 wt % Step 1 & Step 2 similar to sample 1 preparation Step 3 Incubation of heat treated biomass with citrate reduced AgNPs Step 1 Wet biomass synthesis Step 2 Wet biomass incubated with citrate reduced AgNPs Step 3 Final heat treatment. Step 1 1 g of Valcon XC 72 soaked in 20 mL of 1 M AgNO3 for 24 h Step 2 Thorough washing until the washings are free of silver Step 3 Reduction of immobilized silver ions to silver nanoparticles followed by thorough washing
incubating the citrate reduced AgNPs with wet fungal biomass. The wet biomass was incubated with the fresh batches of citrate reduced AgNPs, and the process was continued until the amount of AgNPs in the supernatant remained unchanged. This wet biomass immobilized AgNPs were then heat-treated in inert condition as mentioned above, and this was labeled sample 5. Finally, in order to test our materials against silver immobilized standard carbon materials we further prepared controls with commercially available carbon. The samples were prepared following the report by Bandyopadhyaya et al.9 Typically, 1 g of Valcon XC 72 (Cabot Corporation, Boston, Massachusetts) was vigorously stirred with 20 mL of 1 M AgNO3 for 24 h, followed by washing until the washing was free of silver ions. This silver ion loaded carbon was then dried in a desiccator. Further, these silver ions were reduced with 10 mL of 0.2 M NaBH4. The reaction was left to completion for 24 h followed by excess NaBH4 washing. In the subsequent text, this sample is identified as sample 6. The sample identities and their brief preparation procedures are provided in Table 1 for ready reference. Antibacterial Tests. The antibacterial property of sample 2 and specific role of AgNPs were first assessed against the pure carbon material prepared from the biomass without exposing to AgNO3 (sample 1) and the total control (with out silver or carbon) using the colony count method. It was ensured that the carbon weight taken in sample 1 was the same as that taken in the sample 2 experiment. 800 μL of log phase bacterial culture (OD value approximately 0.6 at 600 nm, subsequently this was subjected to serial dilution with 0.9% saline) was incubated with 200 μL of 8.5 mg sample 2 in 10 mL or with controls. The setup was allowed to incubate in a shaker (200 rpm) at 37 °C. 50 μL of the resultant was taken and plated at intervals of 0.5, 1, 2, 4, 8, and 16 h. The SEM samples of the bacteria before and after reactions were fixed with 2.5% glutraldehyde for 2 h, followed by sequential ethanol washing (with 30%, 50%, 70%, 90% ethanol and twice with 100% ethanol) and centrifuging at 5000 rpm for 10 min. At each washing, the samples were incubated with the alcohol for 15 min. The dehydrated samples were drop-cast on silicon wafer followed by gold coating. The sustainability of antibacterial activity of sample 2 was compared with different controls loaded with silver ions or nanoparticles as detailed below. These controls were as follows: silver ions immobilized on the carbon (sample 3), AgNPs immobilized on the carbon (sample 4), AgNPs immobilized on the wet fungal biomass and then heat treated (sample 5), and Valcon XC 72 immobilized with silver ions followed by their reduction Langmuir 2009, 25(19), 11741–11747
Article with NaBH4 (sample 6). The incubation of samples 2, 3, 4, 5, and 6 with bacteria was preformed as mentioned above. From the simple antibacterial tests, it was found that the complete population was destroyed in 2 h. Consequently, for sustainability tests the bacteria were incubated with each of the above-mentioned controls for 3 h in each cycle. After 3 h of incubation, plating was done with 50 μL of the incubated aliquot. The remaining 950 μL was centrifuged at 2000 rpm for 1 min (this was found to be enough to precipitate the silver loaded carbon samples while leaving the bacterial biomass suspended). This was repeated to make sure all the biomass was removed, and the pellet was collected and resuspended in 200 μL of Milli-Q water. To this redispersed aliquot, fresh bacterial inoculum (800 μL) was added and incubated for 3 h to repeat the plating. This procedure was continued for five cycles of plating to test the sustainable antibacterial property. All the plating was done in triplicate. Silver Leaching Experiments. The immobilization efficiencies of silver ions and AgNPs in different samples were confirmed by sonication. From the above results, samples 2, 4, 5, and 6 was taken for this test. 100 mg of the sample dissolved in 2 mL water was sonicated for 5 min followed by 1000 rpm centrifugation; from this supernatant, a fraction was taken to measure UV intensity at 420 nm that is characteristic for the presence of AgNPs absorbance. This soncation was repeated, and every time, the supernatant intensity was measured. This supernatant fraction was also analyzed with AAS to document the silver content released on each sonication. Antiviral Activity. The antiviral property of sample 2 was tested using the plaque count method with two controls, one the carbon alone and the other total control (without silver or carbon). 80 μL of M13 phage dispersed in SM buffer was incubated with 20 μL of 8.5 mg sample 2 in 10 mL or with controls. The setup was allowed to incubate in a shaker (70 rpm) at 37 °C. Two microliters of the resultant was taken and added to 100 μL of E. coli XL1 - blue competent cells at intervals of 0.5, 1, 2, 4, 8, and 16 h. This setup was allowed to infect the competent cells for 15 min in a water bath maintained at 37 °C. The infected inoculum was then mixed with the top agar and plated. Here, the number of plaques formed will depend on the number of the infected bacteria, and that in turn depends on the amount of virus sustained the sample 2. The experiment was again done in triplicate.
Results and Discussion The methods used to prepare the different samples used in this investigation and their identities are described in the Experimental Section and were briefly described in Table 1. Upon incubation of fungal biomass with silver ions, a brown color was imparted to the biomass, indicating the reduction of silver ions to AgNPs inside the biomass. The intracellular synthesis was confirmed with the 5 min water bath sonication. That the supernatant did not show any absorbance gives credence to the conclusion that the nanoparticles were not entrapped on the surface but intracellularly synthesized. Further, the biomass was dried and grained to powder, and the XRD signal was recorded (curve 1, Figure 1A). The XRD patterns obtained from the as-prepared sample and from sample 2 (curve 2, Figure 1A) showed a number of Bragg reflections at d ≈ 2.36, 2.04, 1.45, and 1.23 A˚, characteristic of the fcc silver.26 The peaks in the heat-treated sample were sharper, indicating an improvement in the crystallinity. The Raman signals of samples 1 (Figure 1C) and 2 (Figure 1B) showed features characteristic of disordered carbon with peaks at ∼1590 cm-1 and ∼1350 cm-1 that are designated as G and D peaks, respectively. The data was (25) (a) M€uhlencoert, E.; Mayer, I.; Zapf, M. W.; Vogel, R. F.; Niessen, L. Eur. J. Plant Pathol. 2004, 110, 651–659. (b) Das, A.; Nanda, G. Lett. Appl. Microbiol. 1995, 20, 141–144. (c) Klechkovskaia, V. V.; Otroshko, T. A.; Egorov, N. S. Mikrobiologiia 1979, 48, 820–825. (26) Compared with JCPDS File No. 040-783.
DOI: 10.1021/la901024p
11743
Article
Vijayakumar and Prasad
Figure 1. (A) XRD spectra of biomass incubated with 1 10-3 M AgNO3 (Curve-1) and Curve-2: biomass incubated with 1 10-3
M AgNO3 heat treated at 600 °C (sample 2). (B) Raman spectrum of biomass challenged with AgNO3 (heat treated at 600 °C) (sample 2). (C) Raman spectrum of heat treated biomass (sample 1).
deconvoluted and fitted to three Gaussian peaks. From the intensity of the peaks at 1365 and 1590 cm-1, the graphite inplane domain sizes (La) value were determined to be 2.90 nm in sample 2 and 0.72 nm in sample 1.27 The increase in La from 0.72 to 2.90 could be ascribed to the presence of metal impurities which were shown to facilitate graphitization.28 However, the inplane size of 2.90 nm in sample 2 still suggested the presence of a highly amorphous and disordered carbon matrix, and such disordered carbon support is expected to provide good accessibility for the reaction media to reach the embedded AgNPs. The SEM and TEM (Figure 2) analysis for the sample 2 was in line, broadly, with the above XRD and Raman analyses. The SEM images (Figure 2A) showed the presence of many sharpedged sheets. One image of such an isolated sheet is displayed in the inset of Figure 2A. Analysis of one such sheet with TEM (Figure 2B) revealed the presence of AgNPs embedded in this sheet. The particles were typically less than 20 nm in size and are present all over the carbon matrix (inset Figure 2B). The graphitic nature predominating the amorphous carbon matrix is shown in Figure 2C. The HRTEM image (Figure 2D) of the AgNPs shows a d spacing of 2.3 A˚ that matched well with the lattice spacing of the (111) plane of silver fcc lattice. The kinetics of the bacterial colony formation over time in sample 2 and the controls with both Gram-negative E. coli and the Gram-positive Bacillus subtilis are given in Table 2 and in Figure S1 (Supporting Information). From these data, it is clear that sample 2 was able to express reasonably good bactericidal property. A slow reduction in the colony count of control (without carbon and silver) was also seen. During the serial dilution of the bacteria, the (27) (a) Prasad, B. L. V.; Sato, H.; Enoki, T.; Hishiyama, Y.; Kaburagi, Y.; Rao, A. M.; Eklund, P. C.; Oshida, K.; Endo, M. Phys. Rev., B 2000, 62, 11209–11218. (b) Andersson, O. E.; Prasad, B. L. V.; Sato, H.; Enoki, T.; Hishiyama, Y.; Kaburagi, Y.; Yoshikawa, M.; Bandow, S. Phys. Rev., B 1998, 58, 16387–16395. (28) Murayama, H.; Maeda, T. Nature 1990, 345, 791–793.
11744 DOI: 10.1021/la901024p
cells were dispersed in the saline (∼0.9% NaCl) alone to arrest the reverse osmosis, and no further nutrient was added. Thus, the slow reduction in the control might be attributed to the lack of nutrient medium for the cells that have already reached log phase. An interesting observation was made when cells were treated with pure carbon matrix obtained by heat treatment of biomass (sample 1) alone. There was an increase in the colony count and stability in the number of colonies after 4 h of incubation with respect to the control experiment. This suggested that the carbon matrix might be acting as a nutrient source. The capacity of the carbonaceous support to act as nutrition bait was supported by CHN analysis, which shows 64.71% C, 2.15% H, and 7.21% N. The C/N ratio is approximately 10:1, which would be luxurious for the growth of microorganisms.29 Supplementing this conclusion was the fact that insoluble carbon is generally used as a carrier in biofertilizers, as nutrient support to the bioorganism initial establishment.30 For the same reason, microbes might be attracted to the carbon support in sample 2 also. However, once the bacteria contact this support with immobilized AgNPs the bactericidal effect is brought out. In general, Bacillus subtilis was found to be more resistant than E. coli. This could be due to the peptidoglycan present in the cell wall of Gram-positive bacteria, which is much thicker than that in the Gram-negative bacteria.31 The SEM study of the E. coli with sample 2 conforms to the antibacterial tests (Figure 3). Figure 3A (29) (a) Thingstad, T. F.; Bellerby, R. G. J.; Bratbak, G.; Boersheim, K. Y.; Egge, J. K.; Heldal, M.; Larsen, A.; Neill, C.; Nejstgaard, J.; Norland, S.; Sandaa, R. A.; Skjoldal, E. F.; Tanaka, T.; Thyrhaug, R.; Topper, B. Nature 2008, 455, 387–389. (b) Demoling, F.; Nilsson, O. L; Baath, E. Soil Biol. Biochem. 2008, 40, 370–379. (c) Horst, W. J.; Schenk, M. K.; B€urkert, A.; Claassen, N.; Flessa, H.; Frommer, W. B.; Goldbach, H.; Olfs, H. W.; Romheld, V.; Sattelmacher, B.; Schmidhalter, U.; Schubert, S.; Wiren, N. V.; Wittenmayer, L. Plant nutrition, Kouno, K.; Chowdhury, M. A. H., Nagaoka, T., Ando, T., Eds. Netherlands, 2001; pp 622-623 (30) Paczkowski, M. W.; Berryhill, D. L. Appl. Environ. Microbiol. 1979, 38, 612–615. (31) Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. J. Biomed. Mater. Res. 2000, 52, 662–668.
Langmuir 2009, 25(19), 11741–11747
Vijayakumar and Prasad
Article
Figure 2. (A) SEM image of heat treated biomass containing intracellularly synthesized AgNPs;sample 2 (scale bar 3 μm) with inset showing the magnified image (scale bar 1 μm). (B) TEM image revealing the presence of AgNPs immobilized on a carbonaceous matrix. The inset in (B) displays magnified portion of this image. (C) Carbon support showing wavy graphitic lines. (D) HRTEM image of one of the AgNPs showing the d spacing corresponding to 111 lattice planes of fcc silver. Table 2. Comparative Bactericidal Activity of the Different Samples Investigateda E. coli (104 colonies)
Bacillus subtilis (104 colonies)
intervals (hours) sample 1 control sample 2 sample 1
control
sample 2
0.5 212 ( 9 165 ( 7 263 ( 13 196 ( 10 16 ( 2 1 195 ( 7 142 ( 6 246 ( 11 151 ( 7 6(1 2 183 ( 8 131 ( 1 250 ( 10 133 ( 6 4 175 ( 7 128 ( 2 204 ( 8 116 ( 5 8 153 ( 6 108 ( 5 201 ( 9 118 ( 6 16 151 ( 7 97 ( 4 176 ( 7 96 ( 4 a Please see Table 1 and text for the details on sample identification.
shows the bacteria before exposure to sample 2. Typical rodshaped structures of bacteria with smooth surfaces were clearly seen. After exposing the bacteria to sample 2 (Figure 3B), they were seen to crowd around the carbanaceous material. After a few minutes of interaction of bacteria with sample 2, no rod-shaped bacteria could be see anywhere in sample (inset Figure 3B). Instead, cell debris formed from the disintegration of bacterial cells could be seen all over. The sustainability tests for sample 2 compared with other samples against E. coli and Bacillus subtilis are given in Tables 3 and 4, respectively. From the results, it is obvious that sample 2 displayed better-sustained bactericidal property. The observation shows that sample 2 is highly antibacterial, as no colonies were present until the fifth cycle, whereas in samples 3 and 4, colonies started appearing after the third and fourth cycles, respectively. This might be because silver ions and the AgNPs attached only to the surface in these samples are prone to leaching as compared to impregnated samples.7 The controls that comes closer in performance to sample 2 are samples 5 and 6. In sample 5, the AgNPs Langmuir 2009, 25(19), 11741–11747
were incubated with wet biomass and were then subjected to heat treatment. It is pertinent to mention here that in sample 5, even though the wet biomass was incubated with preformed AgNPs until saturation for ∼2 days with several batches of citrate reduced AgNPs, it attained only 0.56 wt % of silver uptake as compared to sample 2 where 4 wt % of silver uptake is achieved. This difference in silver wt % necessitated our testing sample 5 against sample 2 in two different conditions. In the first condition, we matched the total weight of silver and carbon taken together with that of sample 2. In this condition, sample 2 performed handsomely better. However, when the amount of silver in sample 5 matches that of sample 2 the bactericidal activity of both samples was comparable (i.e., more total amount of sample 5). This necessity to take a large excess of total sample 5 weight to match the performance of sample 2, the extra time needed, and the cumbersome procedure needed to prepare sample 5 all exemplify the advantages of carbon supported AgNPs prepared via the intracellular route (sample 2) over the sample obtained by treating the biomass with preformed particles and subsequent heat treatment (sample 5). In sample 6, again the silver loading was only ∼0.5 wt %, and hence, we conducted the antibacterial tests under the above-mentioned two conditions, i.e., matching the total weight of carbon with that in sample 2 and matching the weight of silver to that in sample 2 (high total amount of sample). Here, while in the latter case the bactericidal effects were comparable to sample 2 in all respects, the former case performs very poorly compared to sample 2. We also wish to point out that, although this commercial carbon was stirred with almost 1 g of AgNO3 for 1 full day, the silver uptake suggests that only ∼1% of initial silver taken gets immobilized while the rest gets washed away. On the other hand, in our preparation (sample 2) ∼75% of silver gets immobilized, again providing evidence for the utility of the intracellular route presented here. DOI: 10.1021/la901024p
11745
Article
Vijayakumar and Prasad
Figure 3. SEM image showing antibacterial activity of sample 2: (A) Bacteria (E. coli) before treatment; (B) Bacteria immediately after treatment with sample 2; (inset) E. coli after 10 min of reaction with sample 2. Note that no rod-shaped cells are present in this inset indicating the complete destruction of E. coli. Table 3. Comparative Sustained Bactericidal Activity of Different Samples with That of E. coli E. coli (104 colonies)
sample no.
no. of cycles
sample 1
sample 2
sample 3
sample 4
sample 5 (silver content matched with sample 2)
1 2 3 4 5
1 2 3 4 5
230 ( 12 307 ( 15 247 ( 13 213 ( 11 236 ( 12
-
29 ( 2 32 ( 2
19 ( 1
-
sample 5 (total wt matched with sample 2)
sample 6 (silver content matched with sample 2)
sample 6 (total wt matched with sample 2)
9(1 10 ( 1 11 ( 2 12 ( 2 10 ( 1
-
11 ( 1 14 ( 1 14 ( 2 15 ( 1 13 ( 1
Table 4. Comparative Sustained Bactericidal Activity of Different Samples with That of Bacillus subtilisa Bacillus subtilis (104 colonies)
sample no.
no. of cycles
sample 1
sample 2
sample 3
sample 4
1 1 287 ( 14 2 2 278 ( 13 3 3 233 ( 11 4 4 317 ( 16 47 ( 2 5 5 255 ( 13 55 ( 3 21 ( 2 a Please see Table 1 and text for the details on sample identification.
Sustainable and controlled release of the AgNPs from sample 2 was again supported by the UV visible intensity (Supporting Information Figure S2) and the silver concentration (Supporting Information Table S1) in the supernatant after sonication. After the first sonication, sample 2 indeed showed absorbance at 420 nm that may be due to the dispersion of the AgNPs on the surface that had undergone sheer stress while grinding that made them not tightly entangled in carbon. However, in the second and third sonication the absorbance was not significant. Hence, the particles masked in the carbon could be concluded to be intact and available for the continuous bactericidal effect, whereas in sample 4, the particles were removed every time, displaying its susceptibility to washing. As expected, this sample was not sustainable in its bactericidal activity after repeated washing. As found in the sustainable bactericidal activity, sample 5 also showed intactness after the first washing as sample 2, but since the critical loading of AgNPs was not reached, it was not on par with sample 2 in bactericidal activity. Finally, while sample 6 after sonication has not shown any evidence for AgNPs in UV-vis study, the AAS (Supporting Information Table S1) showed a reasonable amount of silver leaching. This again demonstrates that the silver 11746 DOI: 10.1021/la901024p
sample 5 (silver content matched with sample 2)
sample 5 (total wt matched with sample 2)
sample 6 (silver content matched with sample 2)
sample 6 (total wt matched with sample 2)
-
14 ( 2 11 ( 1 13 ( 1 16 ( 3 15 ( 2
-
19 ( 1 18 ( 2 19 ( 3 23 ( 1 22 ( 2
Figure 4. Antiviral kinetics for the sample 2 compared to the control and sample 1. Langmuir 2009, 25(19), 11741–11747
Vijayakumar and Prasad
immobilized via the intracellular route presented in this report withstands physical stress better than other samples. The competence of the carbon supported AgNPs as an antiviral agent is clearly established by the plaque assay results (Figure 4 and Supporting Information Figure S3). Here again, carbon supported silver particles prepared through the intracellular way (sample 2) showed significant reduction in the plaque count compared to the control and biomass calcined carbon (sample 1). It was seen that after half an hour the initial PFU (80) was reduced to a mean PFU of 32 in sample 2 as compared to 78 and 72 PFU in the control and sample 1, respectively. Significantly, in sample 2 the trend continued compared to the control until the end of the antiviral kinetics. The silver action on E. coli XL1 - blue competent cells could be ruled out, since the concentration was too low, confirmed with bacterial colony count.
Conclusion AgNPs immobilized on a carbonaceous support were shown to be good bactericidal agents, and such activity is sustained over several cycles. These AgNPs embedded in a carbonaceous matrix were simply obtained by the heat treatment of intracellular
Langmuir 2009, 25(19), 11741–11747
Article
biogenic nanoparticles. The intracellularlly synthesized nanoparticles have otherwise no applications, unless the nanoparticles are isolated from the matrix. As the carbon matrix seems to be supporting the bacterial colony growth, the present material may possess additional advantages to those already existing in literature. Further, they have also shown activity against virus. Acknowledgment. P.S.V. is thankful to DBT for financial aid. We thank DST - UNANST scheme for partial financial support. Help for obtaining Raman spectra by Prof T. Enoki and Prof. K. Takai and HRTEM analysis from JEOL, Japan, is acknowledged with gratitude. The bacterial cultures offered from NCIM are acknowledged gratefully. We thank Proff Sunil K. Mukherjee for fruitful discussion on nanoparticle effect on virus. SEM analysis by Mr. A. B. Gaikwad is acknowledged with gratitude. Supporting Information Available: Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la901024p
11747
