Superior Antibacterial Activity of Zinc Oxide/Graphene Oxide ...
Supporting Information
Superior Antibacterial Activity of Zinc Oxide/Graphene Oxide
Composites
Originating
from
High
Zinc
Concentration Localized around Bacteria Yan-Wen Wang, † Aoneng Cao, † Yu Jiang, † Xin Zhang, † Jia-Hui Liu, ‡ Yuanfang Liu †,‡
†
and Haifang Wang*,†
Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai
200444, China ‡
Beijing National Laboratory for Molecular Sciences, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871, China *
Author for correspondence:
Tel: +86-21-66138026 Fax: +86-21-66135275 [email protected]
Membrane integrity assay The release of intracellular components is regard as a useful indicator of bacterial membrane integrity. Small ions such as potassium and phosphate tend to leach out first, followed by large molecules such as nucleotides (DNA and RNA). Since nucleotides have strong UV absorbance (OD) at 260 nm, the amount of nucleotides released from bacteria could be determined by measuring OD at 260 nm. This method is easy and widely used (Chen, C. Z.; Cooper, S. L. Interactions between Dendrimer Biocides and Bacterial Membranes. Biomaterials 2002, 23, 3359–3368.). We therefore adopted this method to further confirm the membrane disruption of E. coli after treated with ZnO/GO composites.
Briefly, bacteria prepared were suspended in 0.9% saline solution. In the flasks, the bacteria suspension was mixed with the ZnO/GO-1 or ZnO/GO-2 composite suspension. In the mixture, the concentration of bacteria was 107 CFU/mL, and the concentration of the composite was 5 or 10 μg/mL. The untreated bacteria were used as a control. Aliquots of 0.5 mL were taken out from the flasks at 0, 2, 4, 6, 8, 12, 18 and 24 h. The samples taken out were centrifuged to remove the bacteria and the OD value of the supernatants was measured at 260 nm with the Microplate Reader.
Figure S1. Release of nucleotides from bacteria after exposed to the ZnO/GO composites. The nucleotides were detected by measuring the absorbance at 260 nm with the microplate reader.
Adsorption of zinc ions on GO sheets In order to further investigate the mechanism of antibacterial activities, the adsorption of zinc ions on GO sheets was tested. The identical quantity of GO and Zn2+ as in 10 μg/mL of ZnO/GO-1 was suspended in the LB culture medium (bacterial free) with a final volume of 10 mL, which was as same as that used in bacterial experiment. The mixtures were vibrated in a rotary shaker (150 rpm, 37 °C) for 24 h. After 2 h or 24 h, 1.5 mL of the mixtures were taken out and centrifuge at 41400 g for 15 min to remove GO sheets. The supernatants (1 mL) were kept for digestion. Digestion and ICP-AES measurement were completed according to the methods used in the releasing test.
Cytotoxicity of GO
Figure S2. The cytotoxicity of GO to HeLa cells after 24 h incubation.
Cytotoxicity of ZnO NPs
Figure S3. TEM image of ZnO NPs.
Figure S4. XRD spectrum of ZnO NPs.
Table S1. Physical and chemical properties of ZnO NPs.
sample ZnO NPs
2
purity SSA/m .g >99.9% 8.60
-1
crystal phase hexagonal
dTEM /nm width length dXRD/nm 30±10 100±40 40
The cell viability (mitochondrial function) was evaluated with the same procedure as in the test of cytotoxicity of ZnO/GO composites. ZnO NPs were introduced to cells with a final concentration of 12, 120, 240, 600 μM in culture medium. Cells cultured in the medium without adding ZnO NPs were taken as the control.
Figure S5. The cytotoxicity of ZnO NPs to HeLa cells after 24 h incubation.
Superior Antibacterial Activity of Zinc Oxide/Graphene Oxide
Composites
Originating
from
High
Zinc
Concentration Localized around Bacteria Yan-Wen Wang, † Aoneng Cao, † Yu Jiang, † Xin Zhang, † Jia-Hui Liu, ‡ Yuanfang Liu †,‡
†
and Haifang Wang*,†
Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai
200444, China ‡
Beijing National Laboratory for Molecular Sciences, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871, China *
Author for correspondence:
Tel: +86-21-66138026 Fax: +86-21-66135275 [email protected]
Membrane integrity assay The release of intracellular components is regard as a useful indicator of bacterial membrane integrity. Small ions such as potassium and phosphate tend to leach out first, followed by large molecules such as nucleotides (DNA and RNA). Since nucleotides have strong UV absorbance (OD) at 260 nm, the amount of nucleotides released from bacteria could be determined by measuring OD at 260 nm. This method is easy and widely used (Chen, C. Z.; Cooper, S. L. Interactions between Dendrimer Biocides and Bacterial Membranes. Biomaterials 2002, 23, 3359–3368.). We therefore adopted this method to further confirm the membrane disruption of E. coli after treated with ZnO/GO composites.
Briefly, bacteria prepared were suspended in 0.9% saline solution. In the flasks, the bacteria suspension was mixed with the ZnO/GO-1 or ZnO/GO-2 composite suspension. In the mixture, the concentration of bacteria was 107 CFU/mL, and the concentration of the composite was 5 or 10 μg/mL. The untreated bacteria were used as a control. Aliquots of 0.5 mL were taken out from the flasks at 0, 2, 4, 6, 8, 12, 18 and 24 h. The samples taken out were centrifuged to remove the bacteria and the OD value of the supernatants was measured at 260 nm with the Microplate Reader.
Figure S1. Release of nucleotides from bacteria after exposed to the ZnO/GO composites. The nucleotides were detected by measuring the absorbance at 260 nm with the microplate reader.
Adsorption of zinc ions on GO sheets In order to further investigate the mechanism of antibacterial activities, the adsorption of zinc ions on GO sheets was tested. The identical quantity of GO and Zn2+ as in 10 μg/mL of ZnO/GO-1 was suspended in the LB culture medium (bacterial free) with a final volume of 10 mL, which was as same as that used in bacterial experiment. The mixtures were vibrated in a rotary shaker (150 rpm, 37 °C) for 24 h. After 2 h or 24 h, 1.5 mL of the mixtures were taken out and centrifuge at 41400 g for 15 min to remove GO sheets. The supernatants (1 mL) were kept for digestion. Digestion and ICP-AES measurement were completed according to the methods used in the releasing test.
Cytotoxicity of GO
Figure S2. The cytotoxicity of GO to HeLa cells after 24 h incubation.
Cytotoxicity of ZnO NPs
Figure S3. TEM image of ZnO NPs.
Figure S4. XRD spectrum of ZnO NPs.
Table S1. Physical and chemical properties of ZnO NPs.
sample ZnO NPs
2
purity SSA/m .g >99.9% 8.60
-1
crystal phase hexagonal
dTEM /nm width length dXRD/nm 30±10 100±40 40
The cell viability (mitochondrial function) was evaluated with the same procedure as in the test of cytotoxicity of ZnO/GO composites. ZnO NPs were introduced to cells with a final concentration of 12, 120, 240, 600 μM in culture medium. Cells cultured in the medium without adding ZnO NPs were taken as the control.
Figure S5. The cytotoxicity of ZnO NPs to HeLa cells after 24 h incubation.
