Bacterial Properties of Graphene Oxide

Irina Barbolina

Nanomaterial interaction with cells

Many ways nanomaterials (and graphene in particular)can interact with living cells

Depends on morphology, chemistry, physical

parameters of nanomaterials

Opportunities for applications (drag delivery, antimicrobial)

Possible toxicological issues

Large family of graphene materials

Nanomaterial interaction with cells

Opportunities for applications (drag delivery, antimicrobial)

Many ways nanomaterials (and graphene in particular)can interact with living cells

Depends on morphology, chemistry, physical

parameters of nanomaterials

Graphene OxideWater suspension of oxidised graphene flakes

Thickness: 1-20 monolayers

Size: 30nm-50m

Graphene Oxide ApplicationsWater suspension of oxidised graphene flakes

Thickness: 1-20 monolayers

Size: 30nm-50m

Graphene Oxide

membrane

allows only water

vapour through

courtesy of R. Nair

Graphene Oxide ApplicationsWater suspension of oxidised graphene flakes

Thickness: 1-20 monolayers

Size: 30nm-50m

Antibacterial effects of Graphene Oxide

F. Ahmed, D.F. Rodrigues, J. Hazard. Mater., 256-257, (2013), 33-39.

Large number of publications where antibacterial effect is

found.Some papers where an

absence of such effect is reported.

Bringing new material to the market

In-house DevelopmentTestingEvaluationComparisonCompliance

International Standards Material evaluation based on standard

methods for equivalent material

Material evaluation by literature review

Graphene Oxide

International Standards

Graphene Standards

ISO/NP TR 19733: Matrix of characterization and measurement methods for graphene. Under Development

IEC/CD 62565-3-1: Nanomanufacturing -- Material specifications -- Part 3-1: Graphene -- Blank detail specification. Under development

ISO/NP 80004-13: Nanotechnologies -- Vocabulary -- Part 13: Graphene and other two dimensional materials. Under development

Graphene Oxide

Material evaluation based on standard

methods for equivalent material

New group of materials – no equivalences

Graphene Oxide

Enhancement NeutralAntibacterial

Number of Publications

3 4

23

Material evaluation by literature review

 1 Dellieu, L. et al. Do CVD grown graphene films have antibacterial activity on metallic substrates? Carbon 84, 310-316, (2015).2 He, J. L. et al. Killing dental pathogens Using Antibacterial Graphene Oxide. ACS Appl. Mater. Interfaces 7, 5605-5611, (2015).3 Kurantowicz, N. et al. Interaction of graphene family materials with Listeria monocytogenes and Salmonella enterica. Nanoscale Res. Lett. 10, (2015).4 Li, X., Li, F., Gao, Z. & Fang, L. Toxicology of Graphene Oxide Nanosheets Against Paecilomyces catenlannulatus. Bulletin of environmental contamination and toxicology 95, 25-30, (2015).5 Wu, J., Gao, J. C., Xie, Y. J. & Chen, X. P. Photo-nano-therapy for bactericidal using graphene oxide. Biophotonics and Immune Responses X 9324, (2015).6 Mangadlao, J. D. et al. On the antibacterial mechanism of graphene oxide (GO) Langmuir-Blodgett films. Chem. Commun. 51, 2886-2889, doi:10.1039/c4cc07836e (2015).7 Hui, L. W. et al. Availability of the Basal Planes of Graphene Oxide Determines Whether It Is Antibacterial. ACS Appl. Mater. Interfaces 6, 13183-13190, doi:10.1021/am503070z (2014).8 Chen, J. N. et al. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale 6, 1879-1889, doi:10.1039/c3nr04941h (2014).9 Veerapandian, M., Zhang, L. H., Krishnamoorthy, K. & Yun, K. Surface activation of graphene oxide nanosheets by ultraviolet irradiation for highly efficient anti-bacterials. Nanotechnology 24, doi:10.1088/0957-4484/24/39/395706 (2013).10 Ahmed, F. & Rodrigues, D. F. Investigation of acute effects of graphene oxide on wastewater microbial community: A case study. J. Hazard. Mater. 256, 33-39, doi:10.1016/j.jhazmat.2013.03.064 (2013).11 Chen, J. N., Wang, X. P. & Han, H. Y. A new function of graphene oxide emerges: inactivating phytopathogenic bacterium Xanthomonas oryzae pv. Oryzae. J. Nanopart. Res. 15, doi:10.1007/s11051-013-1658-6 (2013).12 Wang, X. P., Liu, X. Q. & Han, H. Y. Evaluation of antibacterial effects of carbon nanomaterials against copper-resistant Ralstonia solanacearum. Colloid Surf. B-Biointerfaces 103, 136-142, doi:10.1016/j.colsurfb.2012.09.044 (2013).13 Nguyen, T. H. D., Lin, M. S. & Mustapha, A. Toxicity of Graphene Oxide on Intestinal Bacteria and Caco-2 Cells. J. Food Prot. 78, 996-1002, doi:10.4315/0362-028x.jfp-14-463 (2015).14 Wahid, M. H. et al. Microencapsulation of bacterial strains in graphene oxide nano-sheets using vortex fluidics. RSC Adv. 5, 37424-37430, doi:10.1039/c5ra04415d (2015).15 Wang, Y. W. et al. Superior Antibacterial Activity of Zinc Oxide/Graphene Oxide Composites Localized around Bacteria. ACS Appl. Mater. Interfaces 6, 2791-2798, doi:10.1021/am4053317 (2014).16 Tang, J. et al. Graphene Oxide-Silver Nanocomposite As a Highly Effective Antibacterial Agent with Species-Specific Mechanisms. ACS Appl. Mater. Interfaces 5, 3867-3874, doi:10.1021/am4005495 (2013).17 Chen, H. Q. et al. Graphene oxide as an anaerobic membrane scaffold for the enhancement of B. adolescentis proliferation and antagonistic effects against pathogens E-coli and S-aureus. Nanotechnology 25, doi:10.1088/0957-4484/25/16/165101 (2014).18 Wang, D., Wang, G. W., Zhang, G. Q., Xu, X. C. & Yang, F. L. Using graphene oxide to enhance the activity of anammox bacteria for nitrogen removal. Bioresour. Technol. 131, 527-530, doi:10.1016/j.biortech.2013.01.099 (2013). 

Graphene Oxide

In-house DevelopmentTestingEvaluationComparisonCompliance

Effect of GO on microorganisms

Graphene OxidePotentially very promising for applications

Cheap to produce

Easy to control the properties

Water-soluble – compatible with bio-applications

Antibacterial effects of Graphene Oxide

F. Ahmed, D.F. Rodrigues, J. Hazard. Mater., 256-257, (2013), 33-39.

Large number of publications where antibacterial effect is

found.Some papers where an

absence of such effect is reported.

Does it work as antibacterial agent?

Graphene Oxide ProductionHummersmethod:

NaNO3 sodium nitrate

KMnO4

potassium permanganate

H2SO4 sulphuric acid

graphite

Typically suspended in waterpH: <2

Graphite Oxide

Graphene Oxide: Sonication and CentrifugationpH: 2 5

Purification

Commercial Graphene Oxide

Typical parameters of Graphene Oxideobtained from different suppliers

Supplier Flake Size Concentration pH

S1 0.5 30 m 0.2-1.5 mg/ml 4 6S2 1 20 m 10 mg/ml 4 5S3 0.5 30 m 4 mg/ml 2 3S4 0.5 10 m 1-1.5 mg/ml 2 3

Different suppliers Different products

Quality Control – What is important?

Graphene Oxide PurificationCleaning procedure

Chemically clean graphene oxide

GtO 1 2 3 4 5 6 72

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

GO sampleFiltratedH2O

pH

GO flakes size control

Current Methods

Cell viability after exposure to material

Growth parameters in the presence of material

• Turbidity• Colorimetric assay for

enzymes• Plate count • Microscopy: direct count;

Life/Dead staining• Community analysis

Effect of Graphene Oxide on Microorganisms

High OD from GO can give false stimulatory results if not corrected for OD in abiotic control + GO

0 2 4 6 8 10 120.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0 µg/ml

80 µg/ml

160 µg/ml

313 µg/ml

625 µg/ml

F-625 µg/ml

Normalised OD600 (OD test - OD abiotic control)

Time, h

Growth of E.coli in the presence of Graphene Oxide (0-625 µg/ml)

Effect of Graphene Oxide on Microorganisms

0 5 10 15 20 25 30-0.5

0.5

1.5

2.5

3.5

4.5

ControlLarge flakesSmall flakes

Time, h

Normalised OD600 (OD test - OD abiotic control)

OD increase caused by material transformation (reduction)

Growth of E.coli in the presence of Graphene Oxide (50 µg/ml) in Minimum media

Effect of Graphene Oxide on Microorganisms

0 5 10 15 20 25 301E+031E+041E+051E+061E+071E+081E+091E+10

Control

Large flakes

Small flakes

Concentration, CFU/ml

Time, h0 2 4 6 8 10 12 14

1E+031E+041E+051E+061E+071E+081E+091E+10

Control

Small flakes

Large flakes

Concentration, CFU/ml

Time, h

R

Growth of E.coli in Minimum media (M9) and rich media (R) in the presence of Graphene Oxide (50µg/ml)

M9

CONTROL GO

Effect of Graphene Oxide on Microorganisms

Recovery of E.coli from GO solution (1mg/ml) held for 2-4 h at 300C.

CONTROL GO

1E+01

1E+02

1E+03

1E+04

1E+05

0h2h4h

Concentration, CFU/ml

1E+01

1E+02

1E+03

1E+04

1E+05

0h2h4h

Concentration, CFU/ml

H2O S4 S3 S3-F S3-pH S3-W

S1 S2 S3 S4 H2O

Conclusions

• Graphene Oxide is not antibacterial in planktonic culture up to 1 mg/ml

• Bactericidal activity of commercial GO samples is caused by impurities

Further investigations

• Effect of GO on biofilm• Effect on microbial community and mixed populations

Acknowledgements/ Thanks/ Collaborations

Images used: Wikipedia, Rahul Nair, Conrad Gesner

THANK YOU FOR YOUR ATTENTION

Prof. Ian S.RobertsProf. Kostas KostarelosProf. Kostya NovoselovColin WoodsDr. Neus Lozano ValdesDhifaf Jasim