SUPPLEMENTARY INFORMATION for

Graphene oxide can induce in vitro and in vivo

mutagenesis
Yuanyuan Liu1, Yi Luo2, Jing Wu3, Yinsong Wang1,*, Xiaoying Yang1, Rui Yang1,
Baiqi Wang1, Jinrong Yang1 & Ning Zhang1,2,*

Methods
Preparation and characterization of GO.

GO was prepared from purified

natural graphite according to a modified Hummers method S1, following our

previous protocolS2. In detail, graphite powder (1 g), NaNO3 (0.75 g) and KMnO4
(3 g) in concentrated H2SO4 (75 mL) were vigorously stirred at room temperature
for 7 days. On completion of the reaction, 5% H 2SO4 (200 mL) aqueous solution
was added, and the temperature was kept at 98 C for 2 h.

Then, the

temperature was reduced to 60 C, H2O2 (30%, 6 mL) was added and the
reaction was further stirred for 2 h. The above mixture was centrifuged to collect
the bottom product and sequentially washed with 5% H 2SO4/0.5% H2O2 (15
times), 5% HCl solution (5 times), and then washed repeatedly with distilled
water until the pH of the supernatant was neutral. Finally the material was dried
to obtain a loose brown powder.
The content of carboxylic acid groups of GO was determined by acid-base
titration. GO (50 mg) was firstly sonicated in 10 mL NaOH solution (0.1 mol/L)

S1

under argon for 40 min, and the solution was stirred for 48 h. The mixture was
then placed into Visking dialysis tubing (Millipore, molecular weight cutoff 1214
kDa) and dialyzed against deionized water until the dialysate became neutral.
The combined dialysate was condensed using a rotary evaporator and titrated
with HCl solution (0.1 mol/L) to reach the neutral point (pH 7.0), as monitored by
a pH meter (pHS-3C).

This gave the amount of carboxylic acid groups for GO

sample. The amount of carbon in GO is estimated to be 4.15 mmol by assuming

that GO is solely composed of carbon. The same procedure was repeated three
times and the average mole percentage of the carboxylic acid groups on GO was
about 8.1%.
Functional groups of GO were detected by FTIR spectroscopy (Nicolet NEXUS
470-ESP, USA) using KBr pellets.

UV-vis absorbance spectroscopy of GO

dispersed in deionized water at the concentration of 0.06 mg/mL was performed

on a DUR-640 Spectrophotometer (Beckman, USA).

Raman spectrum recorded

on a Renishaw RM1000 with excitation from the 514 nm line of an Arion laser
with a power of about 5 mW.

Powder X-ray diffraction (XRD) pattern was

obtained using a Rigaku D/max-2500 diffractometer (Cu K radiation, =0.15418

nm). For thermogravimetric analysis (TGA), a NETZSCH STA 409PC instrument
was used, and sample was heated in dry nitrogen flow (20 sccm) at a rate of 5 C
min-1.

Results and discussion

S2

The fourier transform infrared (FTIR) spectrum of GO shows the presence of OH

at 3416 cm1, C=O at 1736 cm1, C=C at 1624 cm1, and CO at 1070 cm1 (Fig.
S1a). The Ultra-visible (UV-vis) spectrum shows a maximum absorption peak of
GO at 231 nm, corresponding to the * transitions of aromatic CC bonds in
sp2 hybrid regions, and a shoulder peak at 303 nm, corresponding to n*
transition of the C=O bond in sp 3 hybrid regions (Fig. S1b).

In the Raman

spectrum (Fig. S1c), the D and G bands were clearly observed at 1353 and 1591
cm-1, and the other three weak Raman bands, including 2D, D+G, and 2G,
appeared at 2716, 2912, and 3156 cm-1, respectively.

In the x-ray diffraction

(XRD) spectrum (Fig. S1d), the main diffraction peak appeared at 9.7,
corresponding to a d-spacing of approximately 0.79 nm, which was consistent
with the interlayer spacing of GO sheets. The curves of thermogravimetry (TG)
and derivative thermogravimetry (DTG) are shown in Fig. S1e and S1f. The first
13.5 % weight loss at 77 C due to the evaporation of water molecules and the
following 27.4 % weight loss at 237 C due to the loss of CO, CO 2 and steam
resulted from pyrolysis of the labile oxygen-containing functional groups.
above characterizations were basically consistent with the previous studies.S3-S5

S3

All

Figure S1.

FTIR (a), UV-vis (b), Raman (c), XRD (d), TG (e), and DTG (f)

spectra of GO.

S4

Figure S2. The interaction of GO with genomic DNA and its interference
with DNA replication. (a) GO interacted with genomic DNA. Genomic DNA was
incubated with GO at different concentrations for 2 h and then subjected to gel
electrophoresis (lanes 18: the control and GO treatments at 100, 200, 300, 400,
500, 600 and 700 g/mL). (b) Kinetic analysis of the interaction between GO and
genomic DNA. Genomic DNA was incubated with GO at 600 g/mL for 02 h (lane
18: the control and GO treatments at 10, 20, 40, 60, 80, 100 min, and 2 h). (c)
Impairment of GO on the gene replication by using PCR assay. 50 ng of genomic
GNA was used as templates and the reaction mixture was 25 L. (lane 18: the
control and GO treatments at 100, 200, 400, 600, 800, 1000, 1200 ng/mL).

S5

Figure S3. Inhibition of GO with the replication of PKC gene.

5 ng of

plasmid DNA was used as templates and the reaction mixture was 25 L. (lanes
16: the control and GO treatments at 1, 10, 50, 100, and 200 ng/mL).

Figure S4. Western blot analysis of ATM and Rad51 protein expressions
in MDA-MB-231 cells treated with GO. (lane 13: the control and 1-hour GO
treatments at 10 and 100 g/mL; lane 46: the control and 2-hour GO
treatments at 10 and 100 g/mL)

References

S6

S1. Becerril, H. A., et al. Evaluation of solution-processed reduced graphene oxide

films as transparent conductors. ACS Nano 2, 463470 (2008).
S2. Yang, X. Y., et al. High-efciency loading and controlled release of doxorubicin
hydrochloride on graphene oxide. J. Phys. Chem. C 112, 1755417558 (2008).
S3. Shan, C., et al. Water-soluble graphene covalently functionalized by biocompatible
poly-L-lysine. Langmuir 25, 1203012033 (2009).
S4. Marcano, D. C. et al. Improved synthesis of graphene oxide. ACS Nano 4, 4806
4814 (2010).
S5. Shang J., et al. The Origin of fluorescence from graphene oxide. Scientific Reports
2, 792 (2012).

S7