Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide and ...
Supporting Information
Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide and Reduced Graphene Oxide: Membrane and Oxidative Stress
Shaobin Liu, † Tingying Helen Zeng, ‡ Mario Hofmann, ‡ Ehdi Burcombe, ‡ Jun Wei, § Rongrong Jiang, † Jing Kong, ‡ and Yuan Chen†,* †
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
637459 ‡
Department of Electrical Engineering and Computer Sciences, Massachusetts Institute of Technology,
Cambridge, MA 02139 §
Singapore Institute of Manufacturing Technology, Singapore 638075
*[email protected]
I. Graphite oxide (GtO) preparation
S2
II. Reduced graphene oxide (rGO) preparation
S2
III. Raman spectroscopy
S2
IV. X-ray photoelectron spectroscopy (XPS) analysis of GO
S4
V. Dynamic light scattering (DLS) of dispersions
S6
VI. Cell viability and glutathione oxidation control data
S7
VII. Production of superoxide radical anion (O2•−)
S7
VIII. Comparison of GO and rGO antibacterial activity studies
S8
IX. References
S10
S1
I. GtO preparation1, 2 Two grams of K2S2O8, 2 g of P2O5 and 6 mL of 98% H2SO4 were mixed in a 50 mL beaker, and then heated to 80 oC in water bath. One gram of graphite (Gt) powder (Aldrich, synthetic, < 20 µm) was added into the mixture, and kept at 80 oC for 6 h. Then, the mixture was diluted using distilled water, and filtered through 0.20 µm nylon membrane, followed by thorough washing with water and drying. Afterwards, the as-treated dry Gt powder was added into 92 mL of H2SO4 in ice bath. Fifteen grams of KMnO4 was added slowly with stirring. The mixture was heated to 35 oC under vigorous stirring, and kept for 2 h. Next, 184 mL of water was slowly added; 15 min later, 560 mL of water and 10 mL H2O2 was added. Solid powders were collected by centrifugation from the mixture, and then washed with 1:10 HCl and water. Last, GtO power was suspended in distilled water, and metal ions and acids were removed by dialysis.
II. rGO preparation3 GtO powder (100 mg) and 100 mL of water were loaded in a 250 mL round-bottom flask to yield an inhomogeneous yellow-brown dispersion. This GtO dispersion was sonicated 6 h until it became clear without visible particles to yield GO dispersion. Subsequently, hydrazine hydrate (1 mL, 32 mmol) was added to the GO dispersion, and the dispersion was heated in an oil bath at 100 °C with a watercooled condenser for 24 h. A homogeneous black suspension was obtained. The reduced GO was collected through filtration, and washed with vast amounts of water.
III. Raman spectroscopy Raman spectra were obtained using a laser excitation of 532 nm at a power < 1 mW. Figure S1
S2
shows a representative Raman spectrum of GO samples used in this study after removal of a fluorescent background. Several peaks can be observed, and were assigned to the D, G, Gˊ, D+G and 2Dˊ bands. The G band, resulting from the doubly degenerate zone center E2g mode,4 is located at ~1587 cm-1, which indicates the graphitic nature of the material. The existence of the D band at ~1343 cm-1 is due to the scattering of a zone-boundary phonon with defects. These defects could be caused by covalent bonds of carbon atoms with oxygen or other functional groups.5 The D band is not seen in Raman spectra of defect-free Gt, while a high intensity has been reported in similarly generated GO.
Figure S1. Raman spectrum of GO samples
The intensity ratio of the D and G band (ID/IG) is 1.36 for our GO samples. The broad peaks of G and D bands compared to those of standard graphene sheets imply the creation of defects as well. In addition, there are three Raman bands with weaker but recognized features and intensity, called Gˊ, D + G, and 2Dˊ bands, locating at 2700–3200 cm-1. The Gˊ band at 2700 cm-1 from two phonons with opposite momentum in the highest optical branch near the K (A01 symmetry at K),5 is Raman active for crystalline graphitic materials. It is sensitive to the π band in the graphitic electronic structure. The
S3
combination modes of D + G band at ~2900 cm-1 and the second order 2Dˊ band at ~3174 cm-1 emphasize the high defectiveness of GO samples.6 Dˊ band was not observed in our sample. Our Raman results suggest that the material significantly changed upon exfoliation of Gt flakes to more amorphous and defective GO nanosheets.
IV. XPS analysis of GO The carbon-related chemical component species in GO samples were characterized by high resolution XPS spectrum using a Kratos Axis Ultra DLD (delay line detector) spectrometer equipped with a monochromatic Al Ka X-ray source (1486.69 eV). Figure S2 shows a survey scan (pass energy of 160 eV and step size of 1 eV) for GO samples. Apart from the major elements of carbon and oxygen, there are trace amounts of calcium, sodium, sulfur, and nitrogen. These elements can all be associated with the exfoliation oxidation process. High resolution XPS analysis of carbon components in the GO is shown in Figure S3 (pass energy of 20 eV and step size of 0.1 eV). A binding energy of 284.9 eV indicates the existence of C=C sp2 bonds in the GO sheets; while 286.8 eV results from C-O bonds (epoxy and hydroxyl groups), and a binding energy of 288.7 eV gives the evidence of C=O (carbonyl) group formed during the oxidation. According to the integrated areas, (AC=C/AC-O/AC=O = 19479.0/8662.3/1370.1) the ratios of sp2 –bonded carbon atoms vs. carbon atoms in epoxy and hydroxyl groups and in carbonyl groups in the GO sheets are approximately 7/6/1.7 Thus, the ratio of carbon atoms in the perfect graphene sheet to defects in the lattice is 1 to 1. This high defect density agrees with the result obtained by Raman spectroscopy.
S4
survey/2 4 x 10
FW HM 3.4012 3.1699 3.1942 2.7653 3.1724 3.0684
Area 3259 7. 531 592.511 2305 7. 632 2222 . 008 2648 . 919 1138 . 583
At% 77.14 0.78 18.62 0.62 1.24 1.60
C 1s
35
Po s. 285. 0000 400. 0000 532. 0000 1071 .0000 347. 0000 168. 0000
O 1s
Name C 1s N 1s O 1s Na 1s C a 2p S 2p
40
30
Na 1s
CPS
25
20
N 1s
10
Ca 2p
15
S 2p
5
1000
800
600 Bind ing Energ y (eV)
400
200
Figure S2. XPS survey scan for the elements in the GO sample
C 1s/5 x 10
3
C 1s
18
16
C=C
14
12
CPS
10
8
6
C-O
4
2
C=O
0 291
288
285 Binding Energy (eV)
282
279
Figure S3 Carbon components in the GO nanosheets
S5
V. DLS of Gt, GtO, GO and rGO dispersions
Figure S4. Dynamic light scattering spectra of Gt, GtO, GO and rGO dispersions (all dispersions are at 40 µg·mL-1) Aqueous dispersions of Gt, GtO, GO and rGO were characterized by DLS analysis by a ZetaPALS particle size analyzer (Brookhaven, USA) at the scattering angle θ = 90°. The standard spherical particle models were used in DLS. As shown in Figure S4, after sonication, the nominal effective diameters of particles in Gt, GtO, GO and rGO dispersions are 5.25, 4.42, 0.56, and 2.93 µm, respectively. Because most of graphene-based materials are not spherical particles, the model derived diameters are not their real sizes. DLS results provide a quick indication of their different dispersibility.
S6
VI. Cell viability and GSH oxidation control data
Figure S5. Cell viability (a) and oxidation of glutathione (b) control data after incubation for different periods of time. Isotonic saline solutions without graphene-based materials were used as control in cell viability experiments. Bicarbonate buffer (50 mM at pH 8.6) without graphene-based materials were used as control in GSH oxidation experiments. The control data suggest that our incubation conditions would not either affect the cell viability or cause GSH oxidation.
VII. Production of superoxide radical anion (O2•−) The possibility of superoxide radical anion (O2•−) production was evaluated by monitoring the absorption of XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, Fluka). XTT can be reduced by superoxide radical anion (O2•− ) to form water-soluble XTT-formazan with the maximum absorption at 470 nm. XTT (0.4 mM) were dissolved in phosphate buffered saline (PBS) solution at pH 7.0. GO or rGO dispersion (1 mL) in a PBS buffer (80 µg/mL) was mixed with 1 mL of 0.4 mM XTT. The mixture was incubated in dark for 5 h. Afterwards, the mixture was filtered through a 0.45 µm polyethersulfone filter (Acrodisc® Syringe Filters with Supor® Membrane) to remove GO or
S7
rGO. Filtered solution (250 µL) was then placed in a 96-well plate. The changes in absorbance at 470 nm were monitored on a Benchmark Plus microplate spectrophotometer. In this assay, TiO2 (Degussa, P25, 40 µg·mL-1) dispersion was exposed to a UV light source as a positive control.
Figure S6. Production of superoxide radical anion (O2•−) by GO and rGO dispersions. The O2•− production was monitored during the incubation of XTT (0.2 mM) with Gt, GtO, GO or rGO (40 µg/mL) dispersions at pH 7.0 in dark. Incubation with TiO2 (40 µg/mL) under UV radiation was carried out as a positive control.
VII. Comparison of GO and rGO antibacterial activity studies In our current work, the antibacterial activity of GO dispersion is stronger than that of rGO dispersion, which is similar to the result got by Hu et al..8 However, Akhavan et al.9 reported the antibacterial activity of rGO nanowalls is higher than that of GO nanowalls. As proposed in the main text, the antibacterial activity of graphene based materials may be influenced by two important material
S8
Table S1. GO and rGO antibacterial activity studies
References
Methods
current study
dispersion in saline
Aggregate
Oxidation
Antibacterial
size1
capability2
activity
GO
Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide and Reduced Graphene Oxide: Membrane and Oxidative Stress
Shaobin Liu, † Tingying Helen Zeng, ‡ Mario Hofmann, ‡ Ehdi Burcombe, ‡ Jun Wei, § Rongrong Jiang, † Jing Kong, ‡ and Yuan Chen†,* †
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
637459 ‡
Department of Electrical Engineering and Computer Sciences, Massachusetts Institute of Technology,
Cambridge, MA 02139 §
Singapore Institute of Manufacturing Technology, Singapore 638075
*[email protected]
I. Graphite oxide (GtO) preparation
S2
II. Reduced graphene oxide (rGO) preparation
S2
III. Raman spectroscopy
S2
IV. X-ray photoelectron spectroscopy (XPS) analysis of GO
S4
V. Dynamic light scattering (DLS) of dispersions
S6
VI. Cell viability and glutathione oxidation control data
S7
VII. Production of superoxide radical anion (O2•−)
S7
VIII. Comparison of GO and rGO antibacterial activity studies
S8
IX. References
S10
S1
I. GtO preparation1, 2 Two grams of K2S2O8, 2 g of P2O5 and 6 mL of 98% H2SO4 were mixed in a 50 mL beaker, and then heated to 80 oC in water bath. One gram of graphite (Gt) powder (Aldrich, synthetic, < 20 µm) was added into the mixture, and kept at 80 oC for 6 h. Then, the mixture was diluted using distilled water, and filtered through 0.20 µm nylon membrane, followed by thorough washing with water and drying. Afterwards, the as-treated dry Gt powder was added into 92 mL of H2SO4 in ice bath. Fifteen grams of KMnO4 was added slowly with stirring. The mixture was heated to 35 oC under vigorous stirring, and kept for 2 h. Next, 184 mL of water was slowly added; 15 min later, 560 mL of water and 10 mL H2O2 was added. Solid powders were collected by centrifugation from the mixture, and then washed with 1:10 HCl and water. Last, GtO power was suspended in distilled water, and metal ions and acids were removed by dialysis.
II. rGO preparation3 GtO powder (100 mg) and 100 mL of water were loaded in a 250 mL round-bottom flask to yield an inhomogeneous yellow-brown dispersion. This GtO dispersion was sonicated 6 h until it became clear without visible particles to yield GO dispersion. Subsequently, hydrazine hydrate (1 mL, 32 mmol) was added to the GO dispersion, and the dispersion was heated in an oil bath at 100 °C with a watercooled condenser for 24 h. A homogeneous black suspension was obtained. The reduced GO was collected through filtration, and washed with vast amounts of water.
III. Raman spectroscopy Raman spectra were obtained using a laser excitation of 532 nm at a power < 1 mW. Figure S1
S2
shows a representative Raman spectrum of GO samples used in this study after removal of a fluorescent background. Several peaks can be observed, and were assigned to the D, G, Gˊ, D+G and 2Dˊ bands. The G band, resulting from the doubly degenerate zone center E2g mode,4 is located at ~1587 cm-1, which indicates the graphitic nature of the material. The existence of the D band at ~1343 cm-1 is due to the scattering of a zone-boundary phonon with defects. These defects could be caused by covalent bonds of carbon atoms with oxygen or other functional groups.5 The D band is not seen in Raman spectra of defect-free Gt, while a high intensity has been reported in similarly generated GO.
Figure S1. Raman spectrum of GO samples
The intensity ratio of the D and G band (ID/IG) is 1.36 for our GO samples. The broad peaks of G and D bands compared to those of standard graphene sheets imply the creation of defects as well. In addition, there are three Raman bands with weaker but recognized features and intensity, called Gˊ, D + G, and 2Dˊ bands, locating at 2700–3200 cm-1. The Gˊ band at 2700 cm-1 from two phonons with opposite momentum in the highest optical branch near the K (A01 symmetry at K),5 is Raman active for crystalline graphitic materials. It is sensitive to the π band in the graphitic electronic structure. The
S3
combination modes of D + G band at ~2900 cm-1 and the second order 2Dˊ band at ~3174 cm-1 emphasize the high defectiveness of GO samples.6 Dˊ band was not observed in our sample. Our Raman results suggest that the material significantly changed upon exfoliation of Gt flakes to more amorphous and defective GO nanosheets.
IV. XPS analysis of GO The carbon-related chemical component species in GO samples were characterized by high resolution XPS spectrum using a Kratos Axis Ultra DLD (delay line detector) spectrometer equipped with a monochromatic Al Ka X-ray source (1486.69 eV). Figure S2 shows a survey scan (pass energy of 160 eV and step size of 1 eV) for GO samples. Apart from the major elements of carbon and oxygen, there are trace amounts of calcium, sodium, sulfur, and nitrogen. These elements can all be associated with the exfoliation oxidation process. High resolution XPS analysis of carbon components in the GO is shown in Figure S3 (pass energy of 20 eV and step size of 0.1 eV). A binding energy of 284.9 eV indicates the existence of C=C sp2 bonds in the GO sheets; while 286.8 eV results from C-O bonds (epoxy and hydroxyl groups), and a binding energy of 288.7 eV gives the evidence of C=O (carbonyl) group formed during the oxidation. According to the integrated areas, (AC=C/AC-O/AC=O = 19479.0/8662.3/1370.1) the ratios of sp2 –bonded carbon atoms vs. carbon atoms in epoxy and hydroxyl groups and in carbonyl groups in the GO sheets are approximately 7/6/1.7 Thus, the ratio of carbon atoms in the perfect graphene sheet to defects in the lattice is 1 to 1. This high defect density agrees with the result obtained by Raman spectroscopy.
S4
survey/2 4 x 10
FW HM 3.4012 3.1699 3.1942 2.7653 3.1724 3.0684
Area 3259 7. 531 592.511 2305 7. 632 2222 . 008 2648 . 919 1138 . 583
At% 77.14 0.78 18.62 0.62 1.24 1.60
C 1s
35
Po s. 285. 0000 400. 0000 532. 0000 1071 .0000 347. 0000 168. 0000
O 1s
Name C 1s N 1s O 1s Na 1s C a 2p S 2p
40
30
Na 1s
CPS
25
20
N 1s
10
Ca 2p
15
S 2p
5
1000
800
600 Bind ing Energ y (eV)
400
200
Figure S2. XPS survey scan for the elements in the GO sample
C 1s/5 x 10
3
C 1s
18
16
C=C
14
12
CPS
10
8
6
C-O
4
2
C=O
0 291
288
285 Binding Energy (eV)
282
279
Figure S3 Carbon components in the GO nanosheets
S5
V. DLS of Gt, GtO, GO and rGO dispersions
Figure S4. Dynamic light scattering spectra of Gt, GtO, GO and rGO dispersions (all dispersions are at 40 µg·mL-1) Aqueous dispersions of Gt, GtO, GO and rGO were characterized by DLS analysis by a ZetaPALS particle size analyzer (Brookhaven, USA) at the scattering angle θ = 90°. The standard spherical particle models were used in DLS. As shown in Figure S4, after sonication, the nominal effective diameters of particles in Gt, GtO, GO and rGO dispersions are 5.25, 4.42, 0.56, and 2.93 µm, respectively. Because most of graphene-based materials are not spherical particles, the model derived diameters are not their real sizes. DLS results provide a quick indication of their different dispersibility.
S6
VI. Cell viability and GSH oxidation control data
Figure S5. Cell viability (a) and oxidation of glutathione (b) control data after incubation for different periods of time. Isotonic saline solutions without graphene-based materials were used as control in cell viability experiments. Bicarbonate buffer (50 mM at pH 8.6) without graphene-based materials were used as control in GSH oxidation experiments. The control data suggest that our incubation conditions would not either affect the cell viability or cause GSH oxidation.
VII. Production of superoxide radical anion (O2•−) The possibility of superoxide radical anion (O2•−) production was evaluated by monitoring the absorption of XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, Fluka). XTT can be reduced by superoxide radical anion (O2•− ) to form water-soluble XTT-formazan with the maximum absorption at 470 nm. XTT (0.4 mM) were dissolved in phosphate buffered saline (PBS) solution at pH 7.0. GO or rGO dispersion (1 mL) in a PBS buffer (80 µg/mL) was mixed with 1 mL of 0.4 mM XTT. The mixture was incubated in dark for 5 h. Afterwards, the mixture was filtered through a 0.45 µm polyethersulfone filter (Acrodisc® Syringe Filters with Supor® Membrane) to remove GO or
S7
rGO. Filtered solution (250 µL) was then placed in a 96-well plate. The changes in absorbance at 470 nm were monitored on a Benchmark Plus microplate spectrophotometer. In this assay, TiO2 (Degussa, P25, 40 µg·mL-1) dispersion was exposed to a UV light source as a positive control.
Figure S6. Production of superoxide radical anion (O2•−) by GO and rGO dispersions. The O2•− production was monitored during the incubation of XTT (0.2 mM) with Gt, GtO, GO or rGO (40 µg/mL) dispersions at pH 7.0 in dark. Incubation with TiO2 (40 µg/mL) under UV radiation was carried out as a positive control.
VII. Comparison of GO and rGO antibacterial activity studies In our current work, the antibacterial activity of GO dispersion is stronger than that of rGO dispersion, which is similar to the result got by Hu et al..8 However, Akhavan et al.9 reported the antibacterial activity of rGO nanowalls is higher than that of GO nanowalls. As proposed in the main text, the antibacterial activity of graphene based materials may be influenced by two important material
S8
Table S1. GO and rGO antibacterial activity studies
References
Methods
current study
dispersion in saline
Aggregate
Oxidation
Antibacterial
size1
capability2
activity
GO
