pubs.acs.org/Langmuir © 2009 American Chemical Society
Gold Nanoparticles: Microbial Synthesis and Application in Water Hygiene Management Sujoy K. Das,*,† Akhil R. Das,‡ and Arun K. Guha† †
Department of Biological Chemistry and ‡Polymer Science Unit, Indian Association for the Cultivation of Science, 2A & B, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India
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Received February 17, 2009. Revised Manuscript Received April 20, 2009 A green chemical method to synthesize nanogold-bioconjugate and its eco-friendly promising role to purify contaminated waters has been described. Gold nanoparticles of 10 nm average diameter are produced on the surface of Rhizopus oryzae, a fungal strain, by in situ reduction of chloroauric acid (HAuCl4). The nanogold-bioconjugate (NGBC) showed strong adsorption capacity toward different organophosphorous pesticides. The EDXA study confirms adsorption of pesticides on the conjugate material surface. Morphological changes of the NGBC material after adsorption of organophosphorous pesticides were detected by atomic force micrographs. NGBC shows high antimicrobial activity against several Gram-negative and Gram-positive pathogenic bacteria as well as the yeasts Saccharomyces cerevisiae and Candida albicans. The treatment of microbial cells with NGBC caused rupture of cell membrane as revealed in scanning electron and fluorescence micrographs. These unique characteristics of NGBC have been successfully utilized to obtain potable water free from pathogens and pesticides in a single operation.
Introduction In recent years the synthesis of gold nanoparticles have been the focus of intense interest because of their emerging applications in a number of areas such as bioimaging, biosensors, biolabels, biomedicines, and so forth.1-4 Conventionally gold nanoparticles are usually synthesized by reducing a gold salt (NaAuCl4) with sodium citrate [Na2H(C3H5O(COO)3)] or sodium borohydride (NaBH4) followed by surface modification with suitable capping ligands, occasionally organic solvents, which often raise environmental questions.5-7 Researchers are now concentrating on the biosynthesis of metal nanoparticles using both uni- and multicellular organisms.8-11 The biomimetic and biomineralization procedures for nanoscale material synthesis are expected to yield novel and complex structural entities compared with those obtained by the conventional methods. Very limited reports are available for the synthesis of nanoparticles by microorganisms *Corresponding author. E-mail:
[email protected]; sujoydasiacs@ gmail.com. Fax: +91 33 2473 2805. Phone: +91 33 2473 4971/5904 Ext. 502. (1) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (2) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (3) Salem, A. K.; Searson, P. C.; Leong, K. W. Nat. Mater. 2003, 2, 668–671. (4) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115–2120. (5) Masala, O.; Seshadri, R. Annu. Rev. Mater. Res. 2004, 34, 41–81. (6) Hao, E.; Bailey, R. C.; Schatz, G. C.; Hupp, J. T.; Li, S. Nano Lett. 2004, 4, 327–330. (7) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Ferning, D. G. J. Am. Chem. Soc. 2004, 126, 10076–10084. (8) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13611–13614. (9) Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. J. Phys. Chem. C 2007, 111, 16858–16865. (10) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Ramani, R.; Parischa, R.; Ajayakumar, P. V.; Alam, M.; Sastry, M.; Kumar, R. Angew. Chem., Int. Ed. 2001, 40, 3585–3588. (11) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725–735. (12) Bazylinski, D. A.; Frankel, R. B. Nat. Rev. Microbiol. 2004, 2, 217–230. (13) Labrenz, M.; Druschel, G. K.; Thomsen-Ebert, T.; Gilbert, B.; Welch, S. A.; Kemner, K. M.; Logan, G. A.; Summons, R. E.; De Stasio, G.; Bond, P. L.; Lai, B.; Kelly, S. D.; Banfield, J. F. Science 2000, 290, 1744–1747. (14) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129–1132. (15) Shankar, S. S.; Rai, A.; Ankamwar, B.; Singh, A.; Ahmad, A.; Sastry, M. Nat. Mater. 2004, 3, 482–488.
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functioning consecutively as reducing, capping, and stabilizing agents.9-15 Metal nanoparticles are increasingly being used in different emergent areas;1-4 however, their applications in environmental biotechnology16,17 are much limited. Because of small particle size, large surface-to-volume ratio, and easy anchoring capacity on solid surfaces, researchers are now focusing on nanotechnology-based approaches to meet environmental challenges.18,19 The grave concern for human health due to scarcity of clean water has stimulated research for obtaining pure water free from contaminants such as pesticides and pathogenic organisms. Appropriate hygienic treatment eliminates pathogens from water; nevertheless a few may be present occasionally. The extent of pesticide contamination in water is a matter of great concern because of their potential health hazards and entry into the food chain of humans and animals.20-23 In view of extensive variations in the chemical structures of pesticides, it is hardly possible to find a single method suitable for reducing pesticide concentration of potable water to the permissible limit.24 Conventional practices such as adsorption on activated carbon or different biological materials, ultrafiltration, reverse osmosis or electrochemical treatment suffer from a number of limitations.25-28 These include cost effectiveness, low adsorption capacity or inadequate affinity (16) Zhang, W.-X. J. Nanopart. Res. 2003, 5, 323. (17) Wang, C.-B.; Zhang, W.-X. Environ. Sci. Technol. 1997, 31, 2154–2156. (18) Ichinose, N. Superfine Particle Technology; Springer: Berlin, 1992. (19) Cox, D. M.; Brickman, R. O.; Creegan, K.; Kaldor, A. In Clusters and Cluster-Assembled Materials; Averback, R. S., Bernholc, J., Nelson, D. L., Eds.; Materials Research Society: Warrendale, PA, 1991; p 43. (20) Calaf, G. M.; Roy, D. Int. J. Mol. Med. 2008, 21, 261–268. (21) Zeljezic, D.; Garaj-Vrhovac, V. Chemosphere 2002, 46, 295–303. (22) Hernandez, A. F.; Mackness, B.; Rodrigo, L.; Lopez, O.; Pla, A.; Gil, F.; Durrington, P. N.; Pena, G.; Parron, T.; Serrano, J. L.; Mackness, M. I. Hum. Exp. Toxicol. 2003, 22, 565–574. (23) Perry, M. J.; Venners, S. A.; Barr, D. B.; Xu, X. Reprod. Toxicol. 2007, 23, 113–118. (24) EEC Drinking Water Directive, Official Journal N 229/11, Directive 80/ 778/EEC, 1988. (25) Goodrich, A.; Lykins, W.; Klark, M. J. Environ. Qual. 1991, 20, 707–717. (26) Feleke, Z.; Sakakibara, Y. Water Sci. Technol. 2001, 43, 25–33. (27) Sharma, S. R.; Rathore, H. S.; Ahmed, S. R. Ecotoxicol. Environ. Saf. 1987, 14, 22–29. (28) Lievremont, D.; Seigle-murandi, F.; Benoit-guyod, J. L. Water Res. 1998, 32, 3601–3606.
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toward target toxicant. Research activities are now concentrating on the development of nanotechnology-based methodologies18,19 to overcome these problems. The degradation of a wide variety of aromatic and aliphatic halogenated organic compounds by metal nanoparticles has recently been reported.18,19,29,30 This manuscript describes, for the first time, the microbial synthesis of gold nanoparticles using a native Rhizopus oryzae strain, and application of the generated nanogold-bioconjugate (NGBC) in a single-step removal of some model organophosphorus pesticides from water along with the some microorganisms. We believe that the obtained information will play a vital role toward the development of suitable technology for obtaining safe potable water.
Table 1. Changes with Time in Pesticides and E. coli Concentration in Simulated Water after NGBC Treatment time of incubation malathion parathion chlorpyrifos dimethoate E. coli (min) ( μg/mL) ( μg/mL) ( μg/mL) ( μg/mL) (cell/mL) 10.0 5.0 12.0 8.0 1.2 103 controla 5 5 1.5 4 2 40 1.2 N.D 15 10 1.5 N.Db 30 N.D N.D N.D N.D N.D a Control flask received no biomass. b N.D: not detected.
Experimental Section Materials. Chloroauric acid (HAuCl4 3 3H2O) was purchased from Sigma (USA) and used as received. The pesticides (see Supporting Information Figure S1) were purchased from AccuStandard, Inc., USA (purity 98%). Microbiological media and ingredients were procured from Himedia, India. All the other chemicals and biochemicals were purchased from Merck, Germany. Ultrapure Millipore water (18.2 MΩ) was used as solvent. Methods. Preparation of R. oryzae Mycelia. R. oryzae (MTCC 262) used in this study was maintained and cultivated in potato dextrose (20% potato extract and 2% dextrose) medium as described elsewhere.31 Synthesis of Gold Nanoparticles by Fungal Mycelia. The fungal mycelia (1 g) were added to a 100-mL aqueous solution of HAuCl4 (50-500 mg/L), and the reaction mixture was adjusted to pH 3.0. The mycelial suspension was incubated at 30 °C for 24 h under shaking (120 rpm), while bioreduction of chloroaurate ions (AuCl4-) was monitored visually as well as by UV-visible (UVvis) spectroscopic measurement. The fungal mycelia before and after the reaction with chloroaurate ions were harvested by centrifugation (10 000 rpm for 10 min), dried by lyophilization, and then dispersed in deionized water. UV-vis spectroscopic measurement of the dispersed solution was recorded on a Varian Carry 50 Bio spectrophotometer, wherein the appearance of a peak at 540 nm indicated the formation of gold nanoparticles32,33 on the fungal mycelia. Characterization of Gold Nanoparticles. Infrared spectra of the pristine and AuCl4- treated R. oryzae mycelia were recorded on Shimadzu FTIR spectrometer equipped with highly sensitive pyroelectric detector (DLATGS). The pressed pellets were prepared by grinding the samples with KBr in a mortar at 1:100 ratio and analyzed in the region of 4000-400 cm-1 over 500 scans with a resolution of 2 cm-1. The samples for transmission electron microscopic (TEM) analysis were prepared by drop-casting the gold adsorbed mycelial suspension on a carbon-coated TEM grid. The images were recorded on a high-resolution transmission electron microscope (HRTEM; JEOL JEM 2010) operating at an accelerating voltage of 200 kV equipped with energy dispersive X-ray analysis (EDXA) system. The atomic force microscopic (AFM) study was conducted to record the morphological changes of NGBC due to the adsorption of organophosphorous pesticides. The surface topography of NGBC following the adsorption process was also compared with that of the unadsorbed species. The images were recorded by a multimode atomic (29) Nair, A. S.; Pradeep, T. Curr. Sci. 2003, 84, 1560–1564. (30) Nair, A. S.; Tom, R. T.; Suryanarayanan, V.; Pradeep, T. J. Mater. Chem. 2003, 13, 297–300. (31) Das, S. K.; Bhowal, J.; Das, A. R.; Guha, A. K. Langmuir 2006, 22, 7265– 7272. (32) Gole, A.; Dash, C.; Soman, C.; Sainkar, S. R.; Rao, M.; Sastry, M. Bioconjugate Chem. 2001, 12, 684–690. (33) Rangnekar, A.; Sarma, T. K.; Singh, A. K.; Deka, J.; Ramesh, A.; Chattopadhyay, A. Langmuir 2007, 23, 5700–5706.
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Figure 1. (A) UV-vis spectra of the dispersed solution of goldembedded R. oryzae mycelia. (B) TEM micrograph of R. oryzae mycelia after reaction with aqueous HAuCl4 solution. (C) Highresolution images demonstrate formation of gold nanoparticles (10 nm average diameter) on the surface of mycelia. (D) SAED pattern of gold nanoparticles. force microscope (Veeco Metrology, Autoprobe CP-II, Model No AP0100) at ambient conditions (20 ( 2 °C) using silicon probes (RTESPA-M, Veeco, Santa Barbara, CA) in tapping mode for minimizing the sample damage by the scanning tip. The root-mean-square (rms) roughness of the samples was analyzed using ProScan image processing software. The detailed experimental procedure is given in the Supporting Information. Adsorption Experiments. The lyophilized pristine R. oryzae mycelia or NGBC was added (20 mg) to 25 mL of malathion, parathion, chlorpyrifos, dimethoate, and γ-BHC solutions (500 μg/L, pH 6.0), each taken in separate 100-mL Erlenmeyer flasks. The flasks were incubated at 30 °C (ambient temperature) for 24 h with shaking (120 rpm). At the end of incubation, the biomass was separated by centrifugation at 10 000 rpm for 15 min, and the concentration of pesticide in the supernatant was measured by gas chromatography34 (GC; Hewlett-Packard 6890 series) with nitrogen-phosphorus detector (see Supporting Information for details). Unless stated otherwise, each experiment was performed five times. The optimum pH for adsorption of pesticides was determined by suspending the NGBC (20 mg) in solutions of different pH values (pH 2.0-7.0) containing 500 μg/L pesticide solution. The rates of the pesticide (500 μg/L, pH 6.0) adsorption were followed at regular intervals up to 1 h. The samples were collected from individual flasks; as such, no correction was necessary due to the withdrawal of sampling volume. On completion of adsorption experiments, the NGBC materials were (34) van der Hoff, G. R.; van Zoonen, P. J. Chromatogr. A 1999, 843, 301–322.
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Figure 2. EDXA spectrum of the (A) pristine and (B) gold-embedded R. oryzae mycelia recorded in spot profile mode. thoroughly washed with ultrapure water to remove any unadsorbed pesticides and then analyzed by EDXA, AFM, and Fourier transform infrared (FTIR) spectroscopy as described above. Antimicrobial Activity of the NGBC Material. The NGBC material was dispersed initially in deionized water by sonication, and then antimicrobial activity of the dispersed material in solution was assayed against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella sp., Saccharomyces cerevisiae, and Candida albicans by the cup-plate method.35 The microbicidal activities of NGBC on these organisms were determined by staining with a LIVE/DEAD kit following the manufacture’s instructions (Invitrogen, Carlsbad, CA), and the micrographs were recorded on a fluorescence microscope (Olympus BX-UCB). The alteration of the cell wall morphology following treatment with NGBC was observed using scanning electron microscopy (SEM; JEOL JSM 6700F).
Treatment of Simulated Contaminated Water by NGBC Material. Simulated contaminated water was prepared by adding malathion, parathion, chloropyrifos, and dimethoate along with E. coli to 100 mL sterile distilled water taken in a 250-mL flask to reach a final concentration of 35 μg/L and ∼103 cells/mL, respectively (Table 1). The flasks were gently shaken (100 rpm) at 30 °C for different intervals after addition of 5 mg of NGBC material. After the end of the desired incubation period, nanobioconjugate was separated aseptically by filtration through glass wool. Cell count of E. coli and pesticide concentration in the filtrate were determined by plating on MacConkey agar36 and GC analysis,34 respectively.
Results and Discussion Synthesis of Gold Nanoparticles by R. oryzae Mycelia. Incubation of HAuCl4 solution with R. oryzae mycelia (see Experimental Section) induced gradual color change of the biomass from light yellow to colorless and finally to purple within 24 h, indicating the formation of gold nanoparticles on the mycelial surface.10 The purple mycelia (gold particle immobilized mycelia) were collected by centrifugation (10 000 rpm for 10 min), dried by lyophilization and dispersed in alcohol. The UV-vis spectra of the dispersed solution exhibited absorption maximum at about 540 nm (Figure 1A) due to the surface plasmon resonance (SPR) band of the gold nanoparticles;10,32,33 however, the pristine mycelia showed no such absorption band. Control experiments without biomass remained yellow, indicating that the synthesis of gold nanopartitles was mediated by microbial reduction. With increasing initial concentration of the gold ions, the surface coverage of the gold nanoparticles on the mycelia increases (Supporting Information Figure S3) with concomitant increment of the SPR band at 540 nm (Supporting Information Figure S2) that reached saturation at 500 mg/L HAuCl4 concentration. TEM micrographs demonstrate the (35) Rose, S. B.; Miller, R. E. J. Bacteriol. 1939, 38, 525–537. (36) Gavin, P. J.; Peterson, L. R.; Pasquariello, A. C.; Blackburn, J.; Hamming, M. G.; Kuo, K. J.; Thomson, R. B.Jr. J. Clin. Microbiol. 2004, 42, 1652–1656.
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Figure 3. (A) Adsorption of pesticides on the pristine and NGBC material. (B) Adsorption kinetics of organophosphorous pesticides on NGBC material. Data represent an average of five independent experiments; (SD shown by error bar.
formation of gold nanoparticles on the surface of R. oryzae mycelia (Figure 1B). High-resolution image shows decoration of gold nanoparticles (10 nm average diameter) on the mycelial surface (Figure 1C). The micrograph also demonstrates that as-synthesized gold nanoparticles are well-dispersed with no conspicuous agglomeration and stable even up to 6 months; since the absorption band does not change over this period. This indicates that the mycelial surface acts both as reducing as well as capping agent. Figure 1D depicts the selected area electron diffraction (SAED) pattern obtained from the gold nanoparticles (Figure 1C). The Scherrer ring patterns characteristic of the facecentered cubic (fcc) gold is clearly observed, indicating that the structures seen in the TEM images are nanocrystalline in nature.37 The obtained in situ reduction of gold ions and subsequent formation of gold nanoparticles on the mycelial surface speaks in favor of an environmentally friendly synthetic protocol for the generation and stabilization of metal nanoparticles. EDXA and FTIR spectra of the pristine and HAuCl4-treated R. oryzae were recorded to understand the involvement of the active molecules/groups present on the cell surface as a result of the reduction of AuCl4- ions. HAuCl4-treated R. oryzae shows a strong peak of Au at 2.195 keV, characteristic of gold nanoparticles,38 (37) Ahmad, A.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. Langmuir 2003, 19, 3550–3553. (38) Kumar, A.; Vemula, P. K.; Ajayan, P. M.; John, G. Nat. Mater. 2008, 7, 236–241.
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Figure 4. AFM images (scan area 1.5 μm 1.5 μm) of (A) the pristine NGBC and the (B) malathione-, (C) parathione-, (D) chlorpyrifos-, and (E) dimethoate-adsorbed NGBC material. (F) EDXA spectrum of NGBC material after adsorption of organophosphorous pesticide for 20 min.
along with C, N, and O signatures (Figure 2B), whereas the control mycelia depict the absence of a Au peak (Figure 2A). The C, N, and O signals originate from the biomolecules, and that of Au peaks indicate the formation of gold particles on the mycelial surface.37,39 FTIR spectrum of the protonated pristine mycelia (Supporting Information Figure S4A) shows absorption bands at 1652.9 and 1550 cm-1, corresponding to the amide I and II bands, respectively, of polypeptides/proteins present in the cell surface.37,40-43 The more complex amide III band is located near 1350.0 cm-1. The peak at 1405.3 cm-1 may be assigned to the symmetric stretching of the carboxyl side groups in the amino acid residues of the protein molecules.37,40-43 Following reaction with HAuCl4 solution (Supporting Information Figure S4B), the amide I band appeared at 1664.4 and 1635.5 cm-1, and the amide II band appeared at 1544.9 cm-1. The peak at 1379.0 cm-1 corresponding to amide III appeared at 1371.2 cm-1 in the posttreated material. Furthermore, the absorption band for carboxyl groups present in the pristine mycelia at 1405.3 cm-1 disappeared upon completion of the reduction process. The shifting of absorption peaks at 1033.6 to 1025.1 cm-1 indicates phosphate bond intervention in the interaction process. We have reported earlier31 from zeta potential study that the surface of the R. oryzae mycelia is positively charged at low pH value (3.0). This indicates that the negatively charged AuCl4- ions initially bind to the positively charged cell surface and are subsequently reduced to form gold nanoparticles. It is well-known that proteins can interact with gold nanoparticles either through free amine, carboxyl, or phosphate groups37,44 to stabilize them. Thus, it may be concluded that the gold nanoparticles are synthesized by surface-bound protein molecules that also prevent aggregation. The surface bound protein molecules act both as reducing as well as stabilizing agents. (39) Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. Small 2007, 3, 672–682. (40) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 4th ed.; Wiley: New York, 1981; p 166. (41) Heber, J. R.; Stevenson, R.; Boldman, O. Science 1952, 116, 111–116. (42) Guibal, E.; Roulph, C.; Cloirec, P. Environ. Sci. Technol. 1995, 29, 2496– 2503. (43) Schmitt, J.; Flemming, H. C. Int. Biodeterior. Biodegrad. Sci. 1998, 41, 1–11. (44) Xie, J.; Lee, J. Y.; Wang, D. I. C.; Tin, Y. P. ACS Nano 2007, 1, 429–439.
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Adsorption of Pesticides by NGBC. Removal of pesticides from water bodies using a single method is very difficult because of wide variations in their chemical structures. The adsorption behavior of different organophosphorus pesticides on NGBC material was tested in order to facilitate the eco-friendly removal of pesticides from aqueous solution. The adsorption of organophosphorus pesticides (Supporting Information Figure S1A-D) on NGBC increases significantly to 85-99% from 5-25% corresponding values of pristine mycelia (Figure 3A). Surface coverage of the mycelia with gold nanoparticles increases with increase in gold chloride concentration (Supporting Information Figures S2 and S3) resulting in increase pesticide adsorption (Supporting Information Figure S5) which attains a maximum value when the surface coverage reaches saturation level. However, adsorption of γ-BHC (Supporting Information Figure S1E), an organochlorine pesticide, on both pristine and NGBC material remains almost the same (Figure 3A), indicating that the adsorption of malathion, parathion, dimethoate, and chlorpyrifos on NGBC may occur through chemical interaction involving the sulfur atom of pesticides and the gold atoms of the conjugate materials. Nair et al.45 also reported the chemical interaction of malathion and chlorpyrifos with silver particles. Sulfur groups having higher affinity toward metallic gold for soft-soft interaction46 according to Pearson rule may be responsible for increased adsorption of these pesticides on NGBC.47 The same experiment when carried out at different pH values (2.0-7.0) showed no such effect on the adsorption process (data not shown). Juhasz et al.48 and Prosen et al.49 also reported that adsorption of pesticides was unaffected by pH variation. Hence, the remaining study was carried out at pH 6.0. Adsorption kinetics plays an important role in respect to adsorbate removal by the adsorbent. The rate of adsorption of all organophosphorous pesticides used in the present experiment (45) Nair, A. S.; Pradeep, T. J. Nanosci. Nanotechnol. 2007, 7, 1871–1877. (46) Winter, M. J.; Complexes. In d-Block Chemistry; Oxford University Press: New York, 1994; p 21. (47) Pearson, R. G. Inorg. Chem. 1988, 27, 734–740. (48) Juhasz, A. L.; Smith, E.; Smith, J.; Naidu, R. J. Ind. Microbiol. Biotechnol. 2002, 29, 163–169. (49) Prosen, H.; Troha, A.; Zupancic-Kralj, L. Acta Chim. Slov. 2002, 49, 561– 573.
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Figure 5. AFM images (scan area 1.5 μm 1.5 μm) of NGBC on adsorption of (A) malathione, (B) parathione, (C) chlorpyrifos, and (D) dimethoate at different incubation times.
involving NGBC indicates (Figure 3B) that the adsorption process is very fast reaching equilibrium within 10 min. The fast adsorption rate reflects good accessibility of the binding sites of the NGBC to pesticides, thereby reducing reactor volume and time. The structural and morphological alterations of the NGBC material following the adsorption of organophosphorous pesticides were recorded by AFM imaging.50-53 The micrographs of the control NGBC (Figure 4A) depict decorated gold nanoparticles throughout the surface. However, post-adsorbed species are conspicuously different from that of the control NGBC. The micrographs demonstrate (Figure 4B-E) the formation of conglomerated island-type domains of pesticide molecules (as confirmed by EDXA) on the NGBC material along with the disappearance of gold nanoparticles. The post adsorbed species shows additional peaks of S and P, including C, N, O, and Au in the EDXA spectrum (Figure 4F), indicating pesticides binding to NGBC. The surface roughness (rms) values of the NGBC material increase significantly to 60-75 nm (Supporting Information Table S1) upon pesticides adsorption, corresponding to that (15.5 ( 2.5 nm) of the control (unadsorbed) NBGC. The nucleation and growth process of pesticides on the surface were also investigated through kinetic studies.54,55 Figure 5 shows the representative topographic AFM images of NGBC after adsorption of pesticides at different time intervals. The images depict (50) Campbell, J. F.; Tessmer, I.; Thorp, H. H.; Erie, D. A. J. Am. Chem. Soc. 2008, 130, 10648–10655. (51) Kaminskyj, S. G. W.; Dahms, T. E. S. Micron 2008, 39, 349–361. (52) Ahola, S.; Turon, X.; Osterberg, M.; Laine, J.; Rojas, O. J. Langmuir 2008, 24, 11592-11599. (53) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Langmuir 2004, 20, 11594–11599. (54) Ayranci, E.; Hoda, N. Chemosphere 2005, 60, 1600–1607. (55) van Beinum, W.; Beulke, S.; Brown, C. D. Environ. Sci. Technol. 2006, 40, 494–500.
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Figure 6. Antimicrobial activity of NGBC material against (A) E. coli, (B) P. aeruginosa, (C) B. subtilis, (D) S. aureus, (E) S. cerevisiae, and (F) C. albicans. Cup containing (I) dispersed solution of R. oryzae and (II) NGBC; concentration of bioconjugate in each plate was 50 μg/mL.
that the surface morphology of pesticide-adsorbed NGBC is conspicuously different from that of the pristine one. Following adsorption of pesticides, the gold nanoparticles disappeared (Figure 4A), and a few domain-like structures (as indicated by circles in the micrographs) are formed on the surface soon after initiation of the adsorption process (Figure 5). With increasing time of adsorption, more domain-like structures appeared on the surface; the grain size increased laterally and eventually coalesced. The size of gold nanoparticles is ∼10 nm, whereas the domain size of the pesticides is much higher. The reproducibility of the images was confirmed by recording the images from five independent experiments. The same growth pattern was observed in the case of all the pesticide molecules confirming good Langmuir 2009, 25(14), 8192–8199
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Figure 7. Viability of (A) E. coli, (B) P. aeruginosa, (C) B. subtilis, (D) S. aureus, (E) S. cerevisiae, and (F) C. albicans cells following treatment with NGBC for 30 min. Cells were stained with SYTO 9 and propidium iodide and imaged at 100 magnification using a fluorescence microscope. Green fluorescent cells are representative of live cells, while red fluorescent cells are representative of dead or compromised cells.
adsorption of organophosphorous pesticides on the NGBC. Filho et al.56 postulated that, during adsorption of herbicides (atrazine, imazaquin, metribuzin, and paraquat) on conducting polymer film, the first chain of herbicide molecules initially occupy the free sites, thereafter arranging themselves in small nuclei. As the adsorption process continues, all the available sites on the polymer film are occupied, and the nucleation process tends to saturate; eventually, the initially formed nuclei begin to grow. Lvov and Decher57 also assumed that the adsorption of polyanions occurred in two stages: the initial attachment of certain segments of the chains to the substrate followed by the adsorption of remaining segments. Pellenc et al.58 demonstrated that, during adsorption of lysozyme on mica, coverage of the enzyme on the adsorbent increases proportionately with the duration of adsorption period and finally forms lysozyme clustering at saturation time. Our findings corroborate all these postulations. The sequential changes of surface morphology following the adsorption process thus corroborate the removal of pesticides from aqueous solution with time as depicted in Figure 3B. Antibacterial Activity of NGBC. In order to make potable water free from microbial pathogens, we explored the antimicrobial activity of NGBC. The antimicrobial activity of the dispersed NGBC solution (see the Experimental Section) against P. aeruginosa, E. coli, B. subtilis, S. aureus, Salmonella sp., S. cerevesiae, and C. albicans was tested by the cup-plate method.35 We observed a clear zone of inhibition around the cup (II) in the plate (Figure 6) containing an absorbed dispersed solution of NGBC, indicating the antimicrobial activity of NGBC against these organisms; the control experiment with a dispersed solution of pristine R. oryzae in the cup (I) exhibited no zone of inhibition. The viability of these pathogens following interaction with NGBC was also studied by incubating the organisms with dispersed solution of NGBC for 30 min. Upon completion of the incubation period, the microbial cell suspension was stained using a LIVE/DEAD kit following the manufacturer’s (56) Filho, N. C.; Leite, F.; de, L.; Carvalho, E. R.; Ven^ancio, E. C.; Vaz, C. M. P.; Mattoso, L. H. C. J. Braz. Chem. Soc. 2007, 18, 577–584. (57) Lvov, Y.; Decher, G. Crystallogr. Rep. 1994, 39, 628. (58) Pellenc, D.; Bennett, R. A.; Green, R. J.; Sperrin, M.; Mulheran, P. A. Langmuir 2008, 24, 9648–9655.
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instructions. Figure 7 shows the fluorescent microscopic images of microbial cells following NGBC treatment and after being stained with LIVE (green)/DEAD (red) stains following the manufacturer’s instructions. Exposure of microbial cells to NGBC resulted in a significant decrease in cell viability compared to the control cells. Quantification of the viability of the cells was done by containing live (green) versus dead (red) stains.59 There was ∼90% reduction in cell viability ( p < 0.1) with corresponding increase in the number of red dead cells. This observation exhibits the microbicidal activity of NGBC.59-61 The extent of microbial cell membrane disruption following interaction with NGBC was examined by SEM study. The SEM images of cells exposed to NGBC (Figure 8, middle panel) reveal distinct morphological changes compared to that of the control one (Figure 8, left panel). After incubation with NGBC, the integrity of most of the microbial cells is lost, indicating irreversible cell damage and ultimate cell death. High-resolution images (Figure 8, right panel) indicate that the smooth surface of the control cells changes to an irregular one upon treatment with NGBC. Morphological changes of the cells due to NGBC treatment may be attributed to damage of cellular membrane integrity similar to that reported very recently by Kang et al.62,63 in the case of contact of E. coli cells with carbon nanotubes. Treatment of Simulated Contaminated Water with NGBC. Upon successful removal of organophosphorous pesticides and inactivation of microorganisms in separate experiments, we consider that the NGBC may be used to obtain potable water free from pathogens with pesticide concentrations below the safety level24 in a single operation. We prepared simulated contaminated water (see Experimental Section for details) relevant to the environmental condition (59) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.; Sabo-Attwood, T. L. Nano Lett. 2008, 8, 302–306. (60) Ung-Kyu, C.; Kim, M.-H.; Lee, N.-H. J. Microbiol. Biotechnol. 2007, 17, 1880–1884. (61) Novelli, F.; Recine, M.; Sparatore, F.; Juliano, C. Il Farmaco 1999, 54, 232– 236. (62) Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M. Langmuir 2007, 23, 8670–8673. (63) Kang, S.; Herzberg, M.; Rodrigues, D. F.; Elimelech, M. Langmuir 2008, 24, 6409–6413.
DOI: 10.1021/la900585p
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Figure 8. SEM micrographs of control (left panel) and NGBC-treated (middle panel) (A) E. coli, (B) P. aeruginosa, (C) B. subtilis, (D) S. aureus, and (E) S. cerevisiae cells, indicating morphological changes of microbial cells. Higher magnified images (right panel) of the post-treated cells clearly depict severe membrane damage.
containing E. coli (∼103 cells/mL) and 10 μg/L malathion, 5 μg/L parathion, 12 μg/L chlorpyrifos, and 8 μg/L dimethoate. We added 5 mg of NGBC to 100 mL of this water and incubated with gentle shaking at room temperature (30 °C) for different time intervals. At the end of the desired incubation period, NGBC was separated aseptically by filtration through glass wool, and the cell count of E. coli and pesticide concentration in the filtrate were then determined by plating on MacConkey agar36 and GC analysis,34 respectively. We noted that the concentration of the pesticides (Table 1) and E. coli density (Figure 9B-C) in the treated water fall significantly within 10 min compared with the control (Figure 9A). The pesticides levels decreased (Table 1) below detectable limit (
