Bull Environ Contam Toxicol DOI 10.1007/s00128-015-1499-3
Toxicology of Graphene Oxide Nanosheets Against Paecilomyces catenlannulatus Xiaoyu Li • Fengbo Li • Zhimou Gao Lejin Fang
•
Received: 18 September 2014 / Accepted: 6 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Graphene oxide (GO) nanosheets have been extensively investigated to fabricate the graphene in recent years. The migration of GO nanosheets into the environment could lead to the instability of biological system. In this study, the GO nanosheets were synthesized and were characterized by SEM, high resolution TEM, XRD, Raman, FTIR and XPS techniques. Toxicology testing of GO nanosheets against Paecilomyces catenlannulatus (P. catenlannulatus) was performed by measuring the efflux of cytoplasmic materials of P. catenlannulatus. Approximate 35 % of the bacteria could survive on the surface of GO nanosheets compared to the control sample (*92 %) within 3 h, indicating that GO nanosheets presented significantly antibacterial activities. It was observed that the concentration of RNA in the solution was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell. The results showed that the damage of cell membrane of P. catenlannulatus was attributed to the direct contact of the P. catenlannulatus with the extremely sharp edges of GO nanosheets, which resulted in the P. catenlannulatus inactivation. The less resistant to the damage of cell membrane was observed with increasing of GO concentration and contact time. Keywords Graphene oxide Paecilomyces catenlannulatus Toxicology Membrane stress Oxidative stress X. Li F. Li (&) L. Fang The School of Life Science and Environmental Science, Huangshan University, Huangshan 245041, China e-mail: [email protected] Z. Gao The College of Plant Protection, Anhui Agricultural University, Hefei 230036, China
Graphene oxide (GO) nanosheets, as a precursor for graphene synthesis, are produced by the chemical oxidization of flake graphite (Park and Ruoff 2009; Drever et al. 2010). Owing to the unique physical, chemical, electrical and mechanical properties, GO nanosheets have been extensively applied in the many fields such as material sciences (Meyer et al. 2007; Wu et al. 2009; Bai et al. 2010), biosensors (Mohanty and Berry 2008; Hong et al. 2010; Choi et al. 2010) and environmental cleanup (Zhao et al. 2011; Sun et al. 2012a, b; Romanchuk et al. 2013; Sun et al. 2013). GO suspension can be sufficiently dispersed in aqueous solutions due to the presence of substantial oxygen-containing functional groups. These various oxygencontaining functional groups have been identified with a large number of hydroxyl and epoxy groups on the basal plane and the small amounts of carboxy, carbonyl, phenol, lactone, and quinine at the sheet edges of GO nanosheets (Li et al. 2008; Kim et al. 2010; Eda and Chhowalla 2010). The well-dispersed GO suspension can transform into subsurface environments, which could influence the fate and transport of microorganism. It has been demonstrated that the carbon-based materials present a significant cytotoxicity to cells (Lam et al. 2004; Chen et al. 2006) and human (Manna et al. 2005; Ding et al. 2005; Jia et al. 2005; Magrez et al. 2006; Akhavan and Ghaderi 2010; Mejias Carpio et al. 2012; Zhang et al. 2014; Guo and Mei 2014). Kang et al. (2007, 2008) showed that the bactericidal activity of SWCNTs was much stronger that of MWCNTs. Akhavan and Ghaderi (2010) also found that the reduced graphene oxides were more toxic to the bacteria than the unreduced graphene oxides. It is demonstrated that toxicology mechanisms of carbon-based materials for bacteria includes oxidative stress (Nel et al. 2006; Pulskamp et al. 2007; Narayan et al. 2005) and cutting off intracellular metabolic routes (Kang et al. 2007).
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Paecilomyces catenlannulatus are carnivorous fungi specialized in trapping and digesting nematodes, which exists both species that live inside the nematodes from the beginning and others that catch them mostly with glue traps or in rings, some of with constrict on contact (Li et al. 2013). P. catenlannulatus can be useful in controlling nematodes. P. catenlannulatus can kill harmful nematodes by pathogenesis, causing disease in the nematodes (Li et al. 2014). Therefore, P. catenlannulatus can be used as a bionematicide and promising adsorbents. However, the biological reactivity of P. catenlannulatus is more susceptible to the involvement of human-made nanomaterials that has been transported into subsurface environment. To the authors’ knowledge, biological toxicology of GO nanosheets for P. catenlannulatus has not been investigated. The objectives of this study are (1) to characterize the difference in morphology of P. catenlannulatus in the absence and presence of GO nanosheets; (2) to investigate the effect of concentration of GO nanosheets and contact time on biological reactivity of P. catenlannulatus; and (3) to elucidate the cytotoxicity of GO nanosheets to P. catenlannulatus and concentration of RNA in the phosphate buffer solution of the P. catenlannulatus exposed to the nanowalls.
Materials and Methods The GO nanosheets were synthesized by the oxidation of flake graphite under vigorous stirring conditions (Hummers and Offeman 1958). Briefly, 2.0 g flake graphite and 1.5 g NaNO3 (co-solvent) were added into 150 mL concentrated H2SO4 under vigorous stirring and ice-water bath conditions, then 9.0 g of KMnO4 was slowly added over about 2 h. The suspension was continually stirred for 5 days at room temperature. Then 12 mL H2O2 (30 wt%) was added in the suspension, and the mixture was stirred for 2 h at room temperature. After centrifugation at 23,000 rpm for 60 min, the solid phase was dispersed using vigorous stirring and bath ultrasonication for 30 min at the power of 140 W. The centrifugation and ultrasonication were recycled for several times, and then the sample was rinsed with Milli-Q water until the solution was neutral. The few-layered GO was obtained by freeze-drying it in a vacuum tank overnight. Paecilomyces catenlannulatus (strain 13, culture collection of microbiology laboratory, University of Huangshan, China) was isolated from infected pupae of the lipidopteran T. pityocampa in NE China. Cultures were prepared by adding 2.0 g KH2PO4, 1.0 g NaH2PO4, 1.0 g NH4Cl, 0.2 g MgSO47H2O, 0.01 g FeSO47H2O and 10.0 g glucoses into 1 L DI water, then the mixtures were sterilized at 120°C for 30 min. Induction of proteases was
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carried out in solid media as following procedure: Petri dishes with 0.5 % gelatin and 1 % (w/v) agar in the above salt solution were prepared. Three replicate dishes were inoculated in the centre with a 5 9 5 mm fragment from the edge of a 7-day-old P. catenlannulatus colony. Plates were incubated at 25°C in the dark. For 12 days after inoculation, approximately every second day, the diameters of both colony and halo of gelatin degradation were recorded for each plate. Proteolytic activity was estimated as an index (1-colony diameter/diameter of halo). The SEM images were provided with a Philips CM 200UT scanning electron microscope equipped with Noran Voyager X-ray energy-dispersive spectroscopy. The XRD patterns were mounted on Rigaku D/max-IIB diffractometer with Cu-Ka irradiation operating at 30 kV and 45 mA. The Raman spectra were recorded at 514.5 nm by the LabRam HR Raman spectrometer with Ar? laser. FTIR measurements were conducted using a Perkin–Elmer 100 Fourier transform infrared spectrometer equipped with attenuated total reflection sensor in pressed KBr pellets. Spectra at 1.4 cm-1 resolution were acquired by co-addition of 128 scans within the 400–4000 cm-1 region. The sensor and KBr background spectrum was calibrated before each sample. Spectral manipulation including subtraction of gas compensation (CO2 vapor) and baseline correction were carried out using MAT-LAB 7.4 computing environment. The changes in oxygenated functional groups were characterized by XPS using a hemispherical analyzer equipped with monochromatic Al Ka X-ray source (hm = 1486.6 eV) conducting at a vacuum below 10-7 Pa. The bactericidal activities of GO nanosheets against P. catenlannulatus (as fungi model) were investigated. All samples and glassware were sterilized by autoclaving at 120°C for 30 min before each microbiological experiment. The bacteria were cultured on a nutrient agar plate at 37°C for 24 h. Then, the cultured bacteria were added into 10 mL of saline solution to reach the concentration of bacteria of *108 colony forming units per milliliter (CFU/ mL). Briefly, 100 lL of the diluted bacterial suspension (106 CFU/mL) was spread on surface of the sample under sonication conditions for 60 min at 37°C. The bacteria were washed from the surface of the sample with 5 mL of phosphate buffer solution in the sterilized Petri dish. 100 lL of each bacterial suspension was spread on a nutrient agar plate and incubated at 37°C for 24 h to count the surviving bacterial colonies by using an optical microscope. The total number of the cells forming unit was determined by area based estimation. The each data points were the average value of three separate similar runs. The efflux of RNA was measured by using a NanoDrop ND-1000 spectrophotometer. Briefly, the phosphate buffer solution remaining from the antibacterial test of each sample was diluted and then the solution was centrifuged at
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1000 rpm for 30 min. 50 lL of b-mercaptoethanol was added into the aforementioned supernatant of the solution. The RNA of the bacteria was separated using a RNA purification kit.
Results The morphologies of P. catenlannulatus in the absence and presence of GO nanosheets were illustrated in Fig. 1. As shown in Fig. 1a, one can be seen that multilayer wrinkled GO nanosheets with sharp edges (approximately 500 9 250 9 10 nm) were overlaid with random orientations. This sharp edge can directly contacted with P. catenlannulatus. Figure 1b revealed that the size of P. catenlannulatus (*100 9 50 nm) was observed in terms of optical micrograph. However, the size of P. catenlannulatus (*50 9 50 nm) in the presence GO nanosheets (Fig. 1c) was significantly smaller than that of P. catenlannulatus in the absence of GO nanosheets. Figure 1d showed the high resolution TEM image of P. catenlannulatus in the presence GO nanosheets. The XRD, Raman spectra, FTIR and XPS
Fig. 1 Characterization of GO nanosheets and P. catenlannulatus used in this study. a SEM image of GO nanosheets; b optical microphotograph of P. catenlannulatus; c SEM image of P.
were used to further investigate the changes in the chemical properties of GO. Figure 2a showed the XRD pattern of asprepared GO. The only weak and broad peak at 2h = 10° (d = 0.86 nm) was observed, which was significantly higher than that of graphite (d = 0.34 nm). According to the Raman spectra (Fig. 2b), the D band (at *1350 cm-1) and G band (at *1580 cm-1) were associated with the disordered sp3-hybridized carbon and graphitic sp2 carbon atoms, respectively. Figure 2c showed the FTIR spectra of GO in the presence and absence of P. catenlannulatus. It is observed that the broad peaks of GO nanosheets at *1130, 1507, 1602, 1841 and 3690 cm-1 were assigned to the epoxy, C=C, carboxyl, carbonyl and hydroxyl, respectively (Bagri et al. 2010). However, the shift of oxygenated functional groups of GO ? P. catenlannulatus was observed toward the high wavenumber. As shown, in Fig. 2d, c 1s spectra of GO nanosheets can be deconvoluted into sp2 carbon in aromatic rings (C–C, 284.5 eV), carbonyl (–C=O, 287.0 eV) and carboxyl groups (COOH, 289.2 eV), respectively (Yang et al. 2009). The bacterial toxicology of GO nanosheets for P. catenlannulatus was performed by the batch techniques. P.
catenlannulatus in the presence of GO nanosheets; d high resolution TEM image of GO in the presence of P. catenlannulatus
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Fig. 2 a XRD patterns of GO nanosheets; b Raman spectra of GO in the absence and presence of P. catenlannulatus; c FT-IR spectra of GO in the absence and presence of P. catenlannulatus; d XPS spectra of GO in the absence and presence of P. catenlannulatus
90 80 Servival bacteria RNA
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Fig. 3 Cytotoxicity of GOs to P. catenlannulatus and concentration of RNA in the phosphate buffer solution of the P. catenlannulatus to the GOs
catenlannulatus can be used as appropriate models of fungi to test the bacterial toxicology of GO nanosheets. The bare stainless steel substrate was used as a control sample to
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have a bench mark. Figure 3 showed the bactericidal activity of GO nanosheets against P. catenlannulatus. As shown in Fig. 3, GO nanosheets presented significantly antibacterial activities, approximate 35 % of the bacteria could survive on the surface of GO nanosheets compared to the control sample (*92 %) within 3 h. The toxicology of GO nanosheets through the damage of cell membrane of the bacteria can also be investigated by measuring the intracellular materials in the phosphate buffer solution of the bacteria exposed to the GO nanosheets. The efflux of cytoplasmic materials of the bacteria was determined by measuring the concentration of RNA in the solution (Fig. 3). It was observed that concentration of RNA in the solution of the bacteria exposed to the GO nanosheets was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell.
Discussions The GO were satisfactorily synthesized by the modified Hummers method and were characterized by batch
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characteristic techniques. As demonstrated by SEM, the significant difference in morphology of P. catenlannulatus in the presence and absence of GO nanosheets could be due to the inactivation of P. catenlannulatus with the extremely sharp edges of GO nanosheets. The smaller size of P. catenlannulatus in the presence GO nanosheets was observed, which was consistent with the results reported by Hu et al. who found that GO suspensions inhibited the growth of Escherichia coli bacteria (Hu et al. 2010). As shown in XRD, the only weak and broad peak was observed at 2h = 10° (d = 0.86 nm), which was significantly higher than that of graphite (d = 0.34 nm). The increased c-axis spacing was attributed to the incorporation of massive oxygenated functional groups on the surface of GO nanosheets (Dikin et al. 2007; Sun et al. 2012a, b). It was demonstrated that the D peak comes from the structural imperfections created by the attachment of oxygenated groups on the carbon basal planes (Pimenta et al. 2007; Yang et al. 2009; Chen et al. 2012). The decrease of relative intensity of D band for GO in the presence of P. catenlannulatus could be resulted from the interaction of oxygenated functional groups of GO with P. catenlannulatus. It should be noted that the carbonyl group of GO nanosheets in the presence of P. catenlannulatus was not observed by FTIR spectra, which could be attributed to the formation of complexes between P. catenlannulatus and carbonyl group of GO nanosheets. The characteristic results indicated that the presence of P. catenlannulatus changed the nanostructure and chemical properties of GO nanosheets. The characteristic results indicated that the cell membrane of the bacteria was effectively damaged by direct contact with the very sharp edges of the nanosheets and charge transfer between GO and the bacteria, resulting in more cell membrane damage of the bacteria. The concentration of RNA in the solution of the bacteria exposed to the GO nanosheets was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell. The bacterial cell exhibited slightly negative charge with the edge of the nanosheets as good electron acceptors, which lead to the cell membrance damage of the bacteria. The strong interaction between the more sharpened edges of the nanosheets with the cell membrane of the bacteria resulted in a charge transfer between the bacteria and the edge of the nanosheets. Therefore, the direct contact interaction of the bacteria with the very sharp edge of the nanosheets resulted in more damage to the cell membrance of P. catenlannulatus bacteria. It was determined that the GO nanosheets can be proposed as one of the excellent and ideal nanostructures for an effective direct contact interaction with P. catenlannulatus due to its extremely high aspect ratio.
Based on characteristic results, the GO nanosheets were satisfactorily synthesized by chemical oxidation method. Biological toxicology of GO nanosheets for P. catenlannulatus was performed by measuring its efflux of cytoplasmic materials. The results showed that the damage of cell membrane of the P. catenlannulatus caused by direct contact of the P. catenlannulatus with the extremely sharp edges of GO nanosheets, which resulted in the P. catenlannulatus inactivation. The interaction mechanism between GO nanosheets and P. catenlannulatus was membrane stress and oxidative stress. Acknowledgments Financial supports from 2012 annual national forestry public welfare industry research projects (No. 201304407) are acknowledged.
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Toxicology of Graphene Oxide Nanosheets Against Paecilomyces catenlannulatus Xiaoyu Li • Fengbo Li • Zhimou Gao Lejin Fang
•
Received: 18 September 2014 / Accepted: 6 February 2015 Ó Springer Science+Business Media New York 2015
Abstract Graphene oxide (GO) nanosheets have been extensively investigated to fabricate the graphene in recent years. The migration of GO nanosheets into the environment could lead to the instability of biological system. In this study, the GO nanosheets were synthesized and were characterized by SEM, high resolution TEM, XRD, Raman, FTIR and XPS techniques. Toxicology testing of GO nanosheets against Paecilomyces catenlannulatus (P. catenlannulatus) was performed by measuring the efflux of cytoplasmic materials of P. catenlannulatus. Approximate 35 % of the bacteria could survive on the surface of GO nanosheets compared to the control sample (*92 %) within 3 h, indicating that GO nanosheets presented significantly antibacterial activities. It was observed that the concentration of RNA in the solution was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell. The results showed that the damage of cell membrane of P. catenlannulatus was attributed to the direct contact of the P. catenlannulatus with the extremely sharp edges of GO nanosheets, which resulted in the P. catenlannulatus inactivation. The less resistant to the damage of cell membrane was observed with increasing of GO concentration and contact time. Keywords Graphene oxide Paecilomyces catenlannulatus Toxicology Membrane stress Oxidative stress X. Li F. Li (&) L. Fang The School of Life Science and Environmental Science, Huangshan University, Huangshan 245041, China e-mail: [email protected] Z. Gao The College of Plant Protection, Anhui Agricultural University, Hefei 230036, China
Graphene oxide (GO) nanosheets, as a precursor for graphene synthesis, are produced by the chemical oxidization of flake graphite (Park and Ruoff 2009; Drever et al. 2010). Owing to the unique physical, chemical, electrical and mechanical properties, GO nanosheets have been extensively applied in the many fields such as material sciences (Meyer et al. 2007; Wu et al. 2009; Bai et al. 2010), biosensors (Mohanty and Berry 2008; Hong et al. 2010; Choi et al. 2010) and environmental cleanup (Zhao et al. 2011; Sun et al. 2012a, b; Romanchuk et al. 2013; Sun et al. 2013). GO suspension can be sufficiently dispersed in aqueous solutions due to the presence of substantial oxygen-containing functional groups. These various oxygencontaining functional groups have been identified with a large number of hydroxyl and epoxy groups on the basal plane and the small amounts of carboxy, carbonyl, phenol, lactone, and quinine at the sheet edges of GO nanosheets (Li et al. 2008; Kim et al. 2010; Eda and Chhowalla 2010). The well-dispersed GO suspension can transform into subsurface environments, which could influence the fate and transport of microorganism. It has been demonstrated that the carbon-based materials present a significant cytotoxicity to cells (Lam et al. 2004; Chen et al. 2006) and human (Manna et al. 2005; Ding et al. 2005; Jia et al. 2005; Magrez et al. 2006; Akhavan and Ghaderi 2010; Mejias Carpio et al. 2012; Zhang et al. 2014; Guo and Mei 2014). Kang et al. (2007, 2008) showed that the bactericidal activity of SWCNTs was much stronger that of MWCNTs. Akhavan and Ghaderi (2010) also found that the reduced graphene oxides were more toxic to the bacteria than the unreduced graphene oxides. It is demonstrated that toxicology mechanisms of carbon-based materials for bacteria includes oxidative stress (Nel et al. 2006; Pulskamp et al. 2007; Narayan et al. 2005) and cutting off intracellular metabolic routes (Kang et al. 2007).
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Paecilomyces catenlannulatus are carnivorous fungi specialized in trapping and digesting nematodes, which exists both species that live inside the nematodes from the beginning and others that catch them mostly with glue traps or in rings, some of with constrict on contact (Li et al. 2013). P. catenlannulatus can be useful in controlling nematodes. P. catenlannulatus can kill harmful nematodes by pathogenesis, causing disease in the nematodes (Li et al. 2014). Therefore, P. catenlannulatus can be used as a bionematicide and promising adsorbents. However, the biological reactivity of P. catenlannulatus is more susceptible to the involvement of human-made nanomaterials that has been transported into subsurface environment. To the authors’ knowledge, biological toxicology of GO nanosheets for P. catenlannulatus has not been investigated. The objectives of this study are (1) to characterize the difference in morphology of P. catenlannulatus in the absence and presence of GO nanosheets; (2) to investigate the effect of concentration of GO nanosheets and contact time on biological reactivity of P. catenlannulatus; and (3) to elucidate the cytotoxicity of GO nanosheets to P. catenlannulatus and concentration of RNA in the phosphate buffer solution of the P. catenlannulatus exposed to the nanowalls.
Materials and Methods The GO nanosheets were synthesized by the oxidation of flake graphite under vigorous stirring conditions (Hummers and Offeman 1958). Briefly, 2.0 g flake graphite and 1.5 g NaNO3 (co-solvent) were added into 150 mL concentrated H2SO4 under vigorous stirring and ice-water bath conditions, then 9.0 g of KMnO4 was slowly added over about 2 h. The suspension was continually stirred for 5 days at room temperature. Then 12 mL H2O2 (30 wt%) was added in the suspension, and the mixture was stirred for 2 h at room temperature. After centrifugation at 23,000 rpm for 60 min, the solid phase was dispersed using vigorous stirring and bath ultrasonication for 30 min at the power of 140 W. The centrifugation and ultrasonication were recycled for several times, and then the sample was rinsed with Milli-Q water until the solution was neutral. The few-layered GO was obtained by freeze-drying it in a vacuum tank overnight. Paecilomyces catenlannulatus (strain 13, culture collection of microbiology laboratory, University of Huangshan, China) was isolated from infected pupae of the lipidopteran T. pityocampa in NE China. Cultures were prepared by adding 2.0 g KH2PO4, 1.0 g NaH2PO4, 1.0 g NH4Cl, 0.2 g MgSO47H2O, 0.01 g FeSO47H2O and 10.0 g glucoses into 1 L DI water, then the mixtures were sterilized at 120°C for 30 min. Induction of proteases was
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carried out in solid media as following procedure: Petri dishes with 0.5 % gelatin and 1 % (w/v) agar in the above salt solution were prepared. Three replicate dishes were inoculated in the centre with a 5 9 5 mm fragment from the edge of a 7-day-old P. catenlannulatus colony. Plates were incubated at 25°C in the dark. For 12 days after inoculation, approximately every second day, the diameters of both colony and halo of gelatin degradation were recorded for each plate. Proteolytic activity was estimated as an index (1-colony diameter/diameter of halo). The SEM images were provided with a Philips CM 200UT scanning electron microscope equipped with Noran Voyager X-ray energy-dispersive spectroscopy. The XRD patterns were mounted on Rigaku D/max-IIB diffractometer with Cu-Ka irradiation operating at 30 kV and 45 mA. The Raman spectra were recorded at 514.5 nm by the LabRam HR Raman spectrometer with Ar? laser. FTIR measurements were conducted using a Perkin–Elmer 100 Fourier transform infrared spectrometer equipped with attenuated total reflection sensor in pressed KBr pellets. Spectra at 1.4 cm-1 resolution were acquired by co-addition of 128 scans within the 400–4000 cm-1 region. The sensor and KBr background spectrum was calibrated before each sample. Spectral manipulation including subtraction of gas compensation (CO2 vapor) and baseline correction were carried out using MAT-LAB 7.4 computing environment. The changes in oxygenated functional groups were characterized by XPS using a hemispherical analyzer equipped with monochromatic Al Ka X-ray source (hm = 1486.6 eV) conducting at a vacuum below 10-7 Pa. The bactericidal activities of GO nanosheets against P. catenlannulatus (as fungi model) were investigated. All samples and glassware were sterilized by autoclaving at 120°C for 30 min before each microbiological experiment. The bacteria were cultured on a nutrient agar plate at 37°C for 24 h. Then, the cultured bacteria were added into 10 mL of saline solution to reach the concentration of bacteria of *108 colony forming units per milliliter (CFU/ mL). Briefly, 100 lL of the diluted bacterial suspension (106 CFU/mL) was spread on surface of the sample under sonication conditions for 60 min at 37°C. The bacteria were washed from the surface of the sample with 5 mL of phosphate buffer solution in the sterilized Petri dish. 100 lL of each bacterial suspension was spread on a nutrient agar plate and incubated at 37°C for 24 h to count the surviving bacterial colonies by using an optical microscope. The total number of the cells forming unit was determined by area based estimation. The each data points were the average value of three separate similar runs. The efflux of RNA was measured by using a NanoDrop ND-1000 spectrophotometer. Briefly, the phosphate buffer solution remaining from the antibacterial test of each sample was diluted and then the solution was centrifuged at
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1000 rpm for 30 min. 50 lL of b-mercaptoethanol was added into the aforementioned supernatant of the solution. The RNA of the bacteria was separated using a RNA purification kit.
Results The morphologies of P. catenlannulatus in the absence and presence of GO nanosheets were illustrated in Fig. 1. As shown in Fig. 1a, one can be seen that multilayer wrinkled GO nanosheets with sharp edges (approximately 500 9 250 9 10 nm) were overlaid with random orientations. This sharp edge can directly contacted with P. catenlannulatus. Figure 1b revealed that the size of P. catenlannulatus (*100 9 50 nm) was observed in terms of optical micrograph. However, the size of P. catenlannulatus (*50 9 50 nm) in the presence GO nanosheets (Fig. 1c) was significantly smaller than that of P. catenlannulatus in the absence of GO nanosheets. Figure 1d showed the high resolution TEM image of P. catenlannulatus in the presence GO nanosheets. The XRD, Raman spectra, FTIR and XPS
Fig. 1 Characterization of GO nanosheets and P. catenlannulatus used in this study. a SEM image of GO nanosheets; b optical microphotograph of P. catenlannulatus; c SEM image of P.
were used to further investigate the changes in the chemical properties of GO. Figure 2a showed the XRD pattern of asprepared GO. The only weak and broad peak at 2h = 10° (d = 0.86 nm) was observed, which was significantly higher than that of graphite (d = 0.34 nm). According to the Raman spectra (Fig. 2b), the D band (at *1350 cm-1) and G band (at *1580 cm-1) were associated with the disordered sp3-hybridized carbon and graphitic sp2 carbon atoms, respectively. Figure 2c showed the FTIR spectra of GO in the presence and absence of P. catenlannulatus. It is observed that the broad peaks of GO nanosheets at *1130, 1507, 1602, 1841 and 3690 cm-1 were assigned to the epoxy, C=C, carboxyl, carbonyl and hydroxyl, respectively (Bagri et al. 2010). However, the shift of oxygenated functional groups of GO ? P. catenlannulatus was observed toward the high wavenumber. As shown, in Fig. 2d, c 1s spectra of GO nanosheets can be deconvoluted into sp2 carbon in aromatic rings (C–C, 284.5 eV), carbonyl (–C=O, 287.0 eV) and carboxyl groups (COOH, 289.2 eV), respectively (Yang et al. 2009). The bacterial toxicology of GO nanosheets for P. catenlannulatus was performed by the batch techniques. P.
catenlannulatus in the presence of GO nanosheets; d high resolution TEM image of GO in the presence of P. catenlannulatus
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Fig. 2 a XRD patterns of GO nanosheets; b Raman spectra of GO in the absence and presence of P. catenlannulatus; c FT-IR spectra of GO in the absence and presence of P. catenlannulatus; d XPS spectra of GO in the absence and presence of P. catenlannulatus
90 80 Servival bacteria RNA
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Fig. 3 Cytotoxicity of GOs to P. catenlannulatus and concentration of RNA in the phosphate buffer solution of the P. catenlannulatus to the GOs
catenlannulatus can be used as appropriate models of fungi to test the bacterial toxicology of GO nanosheets. The bare stainless steel substrate was used as a control sample to
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have a bench mark. Figure 3 showed the bactericidal activity of GO nanosheets against P. catenlannulatus. As shown in Fig. 3, GO nanosheets presented significantly antibacterial activities, approximate 35 % of the bacteria could survive on the surface of GO nanosheets compared to the control sample (*92 %) within 3 h. The toxicology of GO nanosheets through the damage of cell membrane of the bacteria can also be investigated by measuring the intracellular materials in the phosphate buffer solution of the bacteria exposed to the GO nanosheets. The efflux of cytoplasmic materials of the bacteria was determined by measuring the concentration of RNA in the solution (Fig. 3). It was observed that concentration of RNA in the solution of the bacteria exposed to the GO nanosheets was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell.
Discussions The GO were satisfactorily synthesized by the modified Hummers method and were characterized by batch
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characteristic techniques. As demonstrated by SEM, the significant difference in morphology of P. catenlannulatus in the presence and absence of GO nanosheets could be due to the inactivation of P. catenlannulatus with the extremely sharp edges of GO nanosheets. The smaller size of P. catenlannulatus in the presence GO nanosheets was observed, which was consistent with the results reported by Hu et al. who found that GO suspensions inhibited the growth of Escherichia coli bacteria (Hu et al. 2010). As shown in XRD, the only weak and broad peak was observed at 2h = 10° (d = 0.86 nm), which was significantly higher than that of graphite (d = 0.34 nm). The increased c-axis spacing was attributed to the incorporation of massive oxygenated functional groups on the surface of GO nanosheets (Dikin et al. 2007; Sun et al. 2012a, b). It was demonstrated that the D peak comes from the structural imperfections created by the attachment of oxygenated groups on the carbon basal planes (Pimenta et al. 2007; Yang et al. 2009; Chen et al. 2012). The decrease of relative intensity of D band for GO in the presence of P. catenlannulatus could be resulted from the interaction of oxygenated functional groups of GO with P. catenlannulatus. It should be noted that the carbonyl group of GO nanosheets in the presence of P. catenlannulatus was not observed by FTIR spectra, which could be attributed to the formation of complexes between P. catenlannulatus and carbonyl group of GO nanosheets. The characteristic results indicated that the presence of P. catenlannulatus changed the nanostructure and chemical properties of GO nanosheets. The characteristic results indicated that the cell membrane of the bacteria was effectively damaged by direct contact with the very sharp edges of the nanosheets and charge transfer between GO and the bacteria, resulting in more cell membrane damage of the bacteria. The concentration of RNA in the solution of the bacteria exposed to the GO nanosheets was obviously higher than that of control sample, which could be due to direct contact of the bacterial cell. The bacterial cell exhibited slightly negative charge with the edge of the nanosheets as good electron acceptors, which lead to the cell membrance damage of the bacteria. The strong interaction between the more sharpened edges of the nanosheets with the cell membrane of the bacteria resulted in a charge transfer between the bacteria and the edge of the nanosheets. Therefore, the direct contact interaction of the bacteria with the very sharp edge of the nanosheets resulted in more damage to the cell membrance of P. catenlannulatus bacteria. It was determined that the GO nanosheets can be proposed as one of the excellent and ideal nanostructures for an effective direct contact interaction with P. catenlannulatus due to its extremely high aspect ratio.
Based on characteristic results, the GO nanosheets were satisfactorily synthesized by chemical oxidation method. Biological toxicology of GO nanosheets for P. catenlannulatus was performed by measuring its efflux of cytoplasmic materials. The results showed that the damage of cell membrane of the P. catenlannulatus caused by direct contact of the P. catenlannulatus with the extremely sharp edges of GO nanosheets, which resulted in the P. catenlannulatus inactivation. The interaction mechanism between GO nanosheets and P. catenlannulatus was membrane stress and oxidative stress. Acknowledgments Financial supports from 2012 annual national forestry public welfare industry research projects (No. 201304407) are acknowledged.
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