and >1 × 10 −3 M cause necrosis. [ 22 ] Thus, given the widespread presence of H 2 O 2 in the body and environment, and the future impact graphene may have on human health and environment, systematic studies need to be performed to investigate the direct chemical interaction between H 2 O 2 and graphene. As a fi rst step, in this communication, we examine the effects of H 2 O 2 on multilayered pristine graphene at physiologically and envi-ronmentally relevant concentrations (1–10 000 × 10 −6 M ). [ 19,22,23 ]
Pristine multilayered graphene deposited on nickel wafer (wafer size = 1 cm × 1 cm, Graphene Supermarket, Calverton, NY, USA) or directly onto TEM grids was placed in petri dishes and treated with H 2 O 2 solution at concentrations of 1 × 10 −6 , 100 × 10 −6 , or 10 000 × 10 −6 M ( n = 4 per group). Graphene incu-bated with distilled water (DI) served as the control. As H 2 O 2 solutions gradually deteriorate, [ 22 ] the solutions were aspirated out every hour and exchanged with fresh solutions. The experi-ments were terminated after 10 or 25 h. At these time points, solutions were completely removed, and graphene substrates were air-dried. Graphene samples before (time t = 0 h) and after H 2 O 2 treatment (time t = 10 and 25 h) were examined with transmission electron microscopy (TEM), atomic force micros-copy (AFM), and confocal Raman spectroscopy (laser excitation of 532 nm).
Figure 1 shows representative TEM images of graphene-coated TEM grids incubated with various concentrations of H 2 O 2 for 10 h. Figure 1 A shows the control graphene sheet incubated with DI water with no holes or defects, and has the basic hexagonal lattice of graphene, possessing 0.142 nm carbon–carbon bond length, occupying ≈0.17 nm 2 area. [ 24 ] Compared with the control group (Figure 1 A), graphene with 1 × 10 −6 M H 2 O 2 treatment after 5 h showed the pres-ence of randomly distributed small holes as seen in Figure 1 B (arrows point to few representative holes). The diameter dis-tribution of holes on graphene was measured by analyzing multiple ( n = 20) TEM images. Incubation of graphene with 1 × 10 −6 M H 2 O 2 for 10 h resulted in the formation of holes ranging from 1 to 15 nm diameter (representing an area up to 175 nm 2 ). Thus, these visible holes indicate a “cluster effect,” i.e., the holes, or defect sites are generated initially by random attack of H 2 O 2 followed by progressive attraction of more H 2 O 2 to destroy carbon–carbon bonds around the ini-tial defect sites. This effect is dependent on the concentration of H 2 O 2 , and enhanced by higher concentrations of H 2 O 2 . Upon treatment with 100 × 10 −6 M H 2 O 2 , formation of larger-sized holes (10–15 nm) was observed (Figure 1 C). Addition-ally, Figure 1 C also shows formation of lighter (few graphene layers) and darker regions (multiple graphene layers) indi-cating the degradation of multilayered graphene. Figure 1 D shows graphene after incubation with 10 000 × 10 −6 M H 2 O 2 . Large-defect sites (10–30 nm) and few graphene layers were observed corresponding to the degradation of majority of the graphene sheets.
W. Xing, G. Lalwani, Prof. B. Sitharaman Department of Biomedical Engineering Stony Brook University Stony Brook , New York 11794–5281, USA E-mail: [email protected] Dr. I. Rusakova Texas Center for Superconductivity and Advanced Materials University of Houston Science Center Houston, Texas 77004–5002 , USA
Graphene, the 2D carbon nanomaterial, is suitable for diverse applications, ranging from electronics and telecommunication, to energy and healthcare. Despite its tremendous technological and commercial prospects, its interactions with biological and environmental constituents still require thorough examination. Here, we report that pristine multilayered graphene degrades in the presence of the naturally occurring, ubiquitous compound hydrogen peroxide (H 2 O 2 ) at physiologically and environmen-tally relevant concentrations (1–10 000 × 10 −6 M ) at various time points (0–25 h).
Graphene, a 2D sheet comprised of sp 2 -hybridized carbon possessing exciting electrical, mechanical, thermal, and optical properties, [ 1 ] has been investigated as enabling components in fuel cells, [ 2,3 ] sensors, [ 2,4 ] photocatalysis, [ 5 ] electronics, [ 6 ] compos-ites, [ 7 ] electrical and optical biosensors, [ 8 ] drug delivery, [ 9 ] tissue engineering, [ 10,11 ] and imaging probes. [ 12 ] Recent reports predict that graphene may overtake carbon nanotubes in commercial applications. [ 13 ] The development of potential graphene-based commercial technologies for applications in material and bio-medical sciences has raised concerns about its short-term and long-term effects on human health and the environment, [ 14 ] and leads to multiple investigations to assess and evaluate these effects. [ 15 ]
An important attribute to be examined while investigating graphene's effect on human health and environment is its bio- and environmental-degradation properties. Several studies have investigated the biodegradation of carbon nanotubes, [ 16,17 ] wherein enzymes such as horseradish peroxidase (HRP) and human myeloperoxidase (hMPO) were employed to catalyze oxidative degradation of these nanomaterials. A recent study has used a similar strategy on graphene oxide, and exploits the catalytic activity of HRP enzyme. [ 18 ] H 2 O 2 is a component of this degradation process.
H 2 O 2 is a naturally occurring, ubiquitous compound and a strong oxidizing agent, found in rain and surface water, and biota. [ 19,20 ] Its concentration in natural water sources has been determined to be 1–7 × 10 −3 M . [ 19,21 ] In normal living cells, diverse cellular pathways synthesize H 2 O 2 in tightly regulated concentrations varying between 1 × 10 −9 and 700 × 10 −9 M . H 2 O 2 steady-state concentrations >1 × 10 −6 M cause oxidative stress,
AFM was performed (using a previously described proce-dure) [ 11 ] to assess the surface topography of graphene sheets before and after H 2 O 2 treatment. Samples were incubated with DI water and 10 000 × 10 −6 M H 2 O 2 for 25 h. Pristine graphene sheets ( Figure 2 A,C,E) appeared smooth, without any topographical defects. Randomly distributed holes in graphene sheets were observed after incubation with H 2 O 2 (Figure 2 B,D,E). The diameter of holes ranged from 5.3 to 13.5 nm. Compared with the height of single graphene sheet (≈0.34 nm), [ 18 ] the depth of the holes (9.4–13.5 nm, inset Figure 2 D) was greater by several folds suggesting the attack of H 2 O 2 on the inner layers of graphene. Once superfi cial defect sites are generated, H 2 O 2 may pass through these defect sites attacking the underlying C–C bonds, and accelerating the deg-radation of graphene at higher concentrations of H 2 O 2 .
Raman spectroscopy was used to characterize the graphene samples before and after treatment with H 2 O 2 at every time point ( Figure 3 ). The ratio of D-band and G-band intensities ( I D / I G ) at all-time points before and after H 2 O 2 treatment are tab-ulated in Table 1 . The D band indicating the degree of disorder in sp 2 -hydridized carbon system was observed between 1335 and 1350 cm −1 , and the G band, indicating stretching of graphitic carbon, was observed between 1570 and 1580 cm −1 . [ 25 ] Table 1 shows decrease in the intensity of D and G bands for all groups treated with various concentration of H 2 O 2 for 25 h, suggesting a gradual structural degradation of graphene. [ 26 ] For groups treated with 1 × 10 −6 and 100 × 10 −6 M H 2 O 2 , the I D / I G ratio increased within 10 h of incubation, and decreased at later time points. Increase in I D / I G ratio during initial time points can be
attributed to increase in the number of defects on graphene, and is consistent with previous reports on enzymatic degradation of graphene. [ 17,18,26 ] The decrease in I D / I G ratio at later time points can be attributed to disintegration of the multiple layers of gra-phene due to progressive increase in the defect sizes, exposing the underlying pristine graphene layers. [ 26 ] The Raman spectra from these underlying pristine graphene layers would have an intense G band, reducing the I D / I G ratio. For the group treated with 10 000 × 10 −6 M H 2 O 2 , I D / I G ratio progressively decreased at all time points, unlike 1 × 10 −6 and 100 × 10 −6 M H 2 O 2 treat-ment groups, which showed an initial increase in the I D / I G ratio within the fi rst 10 h. After 25 h, both D and G bands almost dis-appeared for the 10 000 × 10 −6 M H 2 O 2 treatment group.
G′ band, a characteristic of layered graphene, was observed at ≈2750 cm −1 for all treatment groups. [ 27 ] The intensity of G′ band increased during the fi rst 10 h of H 2 O 2 treatment, decreased, and became negligible at later time points. The G′ band became progressively narrow and down shifted at increasing time points. Several reports have shown the rela-tion of G′ band with the number of layers of graphene in which wider, higher intensity, and an upshift of G′ band corresponds to increasing number of graphene layers. [ 25,27,28 ] In this study, as the degradation progressed, decrease in the width, intensity, and downshift of G′ band corresponds to progressive, layer-by-layer degradation of graphene. A layer-by-layer degradation phenomenon was observed during degradation of multiwalled carbon nanotubes by HRP. [ 17,29 ] Furthermore, differences in G′ band peak intensity and width between various H 2 O 2 treatment groups imply the dependence of graphene degradation rate on
Figure 1. Representative TEM images of multilayered graphene treated with A) deionized water, B) 1 × 10 −6 M H 2 O 2 , C) 100 × 10 −6 M H 2 O 2 , and D) 10 000 × 10 −6 M H 2 O 2 for 10 h. Arrows in (B) indicate the formation of holes on graphene sheets, and arrows in (C) indicate the formation of lighter (few graphene layers) and darker regions (multiple graphene layers) suggesting the degradation of multilayered graphene. It should be noted that the arrows in (B–D) only point to a few representative holes.
the concentration of hydrogen peroxide, i.e., graphene treated with 1 × 10 −6 M H 2 O 2 may degrade at a slower rate compared to treatment with 10 000 × 10 −6 M H 2 O 2 .
The above results taken together clearly indicate the deg-radation of multilayered pristine graphene at a wide range of
H 2 O 2 concentrations found in living systems and environment. Previous studies report that metal ions such as nickel (Ni), iron (Fe), and copper (Cu) can catalyze the degradation of H 2 O 2 to form reactive hydroxyl radicals via the Haber–Weiss reaction. [ 30 ] Ni, Fe, or Cu-based substrates or catalysts are used for graphene
Figure 2. Representative AFM images of multilayered graphene on Ni wafer. A,C,E) are topographical scans of pristine graphene samples (incubated with DI water) and B,D,E) are graphene after 25 h of H 2 O 2 treatment (10 000 × 10 −6 M). Insets in images C and D correspond to the line height profi le. Images E and F are 3D representations of images C and D, respectively.
synthesis using the chemical vapor deposition method. [ 31 ] Thus, in this study, the presence of trace amounts of Ni used during the CVD synthesis of the graphene may be catalyzing the deg-radation of graphene during H 2 O 2 treatment. The results also indicate that rate of degradation of graphene is dependent on H 2 O 2 exposure concentration; increase in concentration
accelerates graphene’s degradation. The visible holes indicate a “cluster effect,” i.e., the holes, or defect sites are generated ini-tially by random attack of H 2 O 2 followed by progressive attrac-tion of more H 2 O 2 to destroy the carbon–carbon bond around initial defect sites. Furthermore, reaction temperature may also infl uence the degradation rate. Additional studies are necessary
Figure 3. Representative Raman spectra of graphene treated with A) 10 000 × 10 −6 M H 2 O 2 , B) 100 × 10 −6 M H 2 O 2 , and C) 1 × 10 −6 M H 2 O 2 for a period of 0, 10, and 25 h. Decrease in the D and G band intensities can be observed after 25 h. Figures D–F show the corresponding I D / I G intensity ratio at every time point .
Table 1. I D / I G ratio of multilayered graphene samples before and after incubation with H 2 O 2 for 25 h.
H 2 O 2 incubation concentration
I D / I G ratio
Before incubation After incubation (25 h)
10 000 × 10 −6 M 0.2312 0.1706
100 × 10 −6 M 0.2312 0.1519
1 × 10 −6 M 0.2312 0.1499
Received: September 25, 2013 Revised: December 12, 2013
Published online:
[1] a) S. Latil , L. Henrard , Phys. Rev. Lett. 2006 , 97 , 036803 ; b) K. S. Novoselov , A. K. Geim , S. V. Morozov , D. Jiang , Y. Zhang , S. V. Dubonos , I. V. Grigorieva , A. A. Firsov , Science 2004 , 306 , 666 ; c) R. F. Service , Science 2009 , 324 , 875 ; d) A. A. Balandin , S. Ghosh , W. Bao , I. Calizo , D. Teweldebrhan , F. Miao , C. N. Lau , Nano Lett. 2008 , 8 , 902 ; e) Q. Bao , H. Zhang , Y. Wang , Z. Ni , Y. Yan , Z. X. Shen , K. P. Loh , D. Y. Tang , Adv. Funct. Mater. 2009 , 19 , 3077 ; f) Q. Bao , K. P. Loh , ACS Nano 2012 , 6 , 3677 .
[2] D. R. Kauffman , A. Star , Analyst 2010 , 135 , 2790 . [3] C. Liu , S. Alwarappan , Z. Chen , X. Kong , C. Z. Li , Biosens. Bioelec-
tron. 2010 , 25 , 1829 . [4] C. H. Lu , H. H. Yang , C. L. Zhu , X. Chen , G. N. Chen , Angew. Chem
Int. Ed. Engl. 2009 , 48 , 4785 . [5] a) N. Zhang , Y. Zhang , Y. J. Xu , Nanoscale 2012 , 4 , 5792 ;
b) Y. Zhang , Z. R. Tang , X. Fu , Y. J. Xu , ACS Nano 2010 , 4 , 7303 ; c) Y. Zhang , N. Zhang , Z. R. Tang , Y. J. Xu , ACS Nano 2012 , 6 , 9777 ; d) J. G. Radich , P. V. Kamat , ACS Nano 2013 , 7 , 5546 .
[6] a) M. Freitag , Nat. Nanotechnol. 2008 , 3 , 455 ; b) R. M. Westervelt , Science 2008 , 320 , 324 .
[7] a) H. Bai , C. Li , X. Wang , G. Shi , Chem. Commun. 2010 , 46 , 2376 ; b) L. Ren , X. Qi , Y. Liu , Z. Huang , X. Wei , J. Li , L. Yang , J. Zhong , J. Mater. Chem. 2012 , 22 , 11765 .
[8] a) H. Jang , Y. K. Kim , H. M. Kwon , W. S. Yeo , D. E. Kim , D. H. Min , Angew. Chem Int. Ed. Engl. 2010 , 49 , 5703 ; b) M. Zhou , Y. Zhai , S. Dong , Anal. Chem. 2009 , 81 , 5603 .
[9] a) X. Sun , Z. Liu , K. Welsher , J. T. Robinson , A. Goodwin , S. Zaric , H. Dai , Nano Res. 2008 , 1 , 203 ; b) S. M. Chowdhury , G. Lalwani , K. Zhang , J. Y. Yang , K. Neville , B. Sitharaman , Biomaterials 2013 , 34 , 283 .
[10] a) T. R. Nayak , H. Andersen , V. S. Makam , C. Khaw , S. Bae , X. Xu , P. L. Ee , J. H. Ahn , B. H. Hong , G. Pastorin , B. Ozyilmaz , ACS Nano 2011 , 5 , 4670 ; b) G. Lalwani , A. T. Kwaczala , S. Kanakia , S. C. Patel , S. Judex , B. Sitharaman , Carbon N Y 2013 , 53 , 90 .
[11] G. Lalwani , A. M. Henslee , B. Farshid , L. Lin , F. K. Kasper , Y. X. Qin , A. G. Mikos , B. Sitharaman , Biomacromolecules 2013 , 14 , 900 .
[12] a) S. Zhu , J. Zhang , C. Qiao , S. Tang , Y. Li , W. Yuan , B. Li , L. Tian , F. Liu , R. Hu , H. Gao , H. Wei , H. Zhang , H. Sun , B. Yang , Chem. Commun. 2011 , 47 , 6858 ; b) B. S. Paratala , B. D. Jacobson , S. Kanakia , L. D. Francis , B. Sitharaman , PLoS One 2012 , 7 , e38185 ; c) G. Lalwani , X. Cai , L. Nie , L. V. Wang , B. Sitharaman , Photoacous-tics 2013 , 1 , 62 ; d) S. Kanakia , J. D. Toussaint , S. M. Chowdhury , G. Lalwani , T. Tembulkar , T. Button , K. R. Shroyer , W. Moore , B. Sitharaman , Int. J. Nanomed. 2013 , 8 , 2821 .
[14] A. D. Maynard , R. J. Aitken , T. Butz , V. Colvin , K. Donaldson , G. Oberdorster , M. A. Philbert , J. Ryan , A. Seaton , V. Stone , S. S. Tinkle , L. Tran , N. J. Walker , D. B. Warheit , Nature 2006 , 444 , 267 .
[15] a) S. Zhang , K. Yang , L. Feng , Z. Liu , Carbon 2011 , 49 , 4040 ; b) Y. Zhang , S. F. Ali , E. Dervishi , Y. Xu , Z. Li , D. Casciano , A. S. Biris , ACS Nano 2010 , 4 , 3181 ; c) S. R. Ryoo , Y. K. Kim , M. H. Kim , D. H. Min , ACS Nano 2010 , 4 , 6587 ; d) B. Parvin , I. Refi , F. Bunshi , Carbon 2011 , 49 , 3907 ; e) T. S. Sreeprasad , T. Pradeep , Int. J. Mod. Phys. B 2012 , 26 , 1242001 .
[16] a) B. L. Allen , G. P. Kotchey , Y. Chen , N. V. K. Yanamala , J. Klein-Seetharaman , V. E. Kagan , A. Star , J. Am. Chem. Soc. 2009 , 131 , 17194 ; b) B. L. Allen , P. D. Kichambare , P. Gou , I. I. Vlasova , A. A. Kapralov , N. Konduru , V. E. Kagan , A. Star , Nano Lett. 2008 , 8 , 3899 ; c) V. E. Kagan , N. V. Konduru , W. Feng , B. L. Allen , J. Conroy , Y. Volkov , I. I. Vlasova , N. A. Belikova , N. Yanamala , A. Kapralov , Y. Y. Tyurina , J. Shi , E. R. Kisin , A. R. Murray , J. Franks , D. Stolz , P. Gou , J. Klein-Seetharaman , B. Fadeel , A. Star , A. A. Shvedova , Nat. Nanotechnol. 2010 , 5 , 354 .
[17] Y. Zhao , B. L. Allen , A. Star , J. Phys. Chem. A 2011 , 115 , 9536 .
and currently underway to test the above hypothesis and pro-vide better understanding of chemical mechanism of H 2 O 2 -mediated degradation of graphene (interplay of H 2 O 2 concen-tration, reaction temperature, and presence of metal ions).
A wide variety of catalytic peroxidase enzymes such as MPO, HRP, and lignin peroxidase (LiP) are present in the body or environment. Several studies have investigated the effects of enzymes such as HRP and hMPO on the degradation of single- and multiwalled carbon nanotubes. [ 16,17 ] A recent study dem-onstrated the degradation of graphene oxide in the presence of HRP. [ 18 ] These studies indicate that enzymatic degradation depends on the surface functionalization of carbon nanomate-rials, and requires pretreatment steps, which include exposure to strong acids and oxidants prior to enzymatic degradation. Additionally, this degradation process has been reported to be effective upon continuous exposure of carbon nanomaterials to enzyme and H 2 O 2 . Thus, peroxidase enzymes could certainly accelerate degradation of graphene in the presence of H 2 O 2 . [ 18 ] However, these enzymes are mainly found in the proximity of certain animal cells (MPO is secreted by infl ammatory cells—neutrophils), plant cells (HRP is present in the root of horse-radish plant), or fungi (LiP is found fungi such as Phanerochaete chrysosporium ). H 2 O 2 , on the other hand, is not restricted to these bio-organisms but is ubiquitous in eukaryotic cells, [ 22 ] and natural resources (fresh and sea water). [ 32 ] Our results strongly suggest that alternative degradation mechanism in the presence H 2 O 2 at physiologically and environmentally relevant concentrations (1–10 000 × 10 −6 M ) could initiate and degrade single- and multilayered pristine graphene. The limitations of the above study are: (1) while concentrations of H 2 O 2 mimic those found in physiological and environmental conditions, the controlled experimental conditions allow all the available H 2 O 2 to interact with graphene; other competing redox processes in biological systems or environment that require H 2 O 2 were absent. The presence of these competing processes will impair the degradation rate. (2) The studies were performed on multi-layered sheets of pristine graphene. Thus, this study only pro-vides insights into the surface degradation effect of H 2 O 2 . Bulk degradation on macroscopic amount of graphene aggregates in the presence of H 2 O 2 still needs to be determined.
Acknowledgements This work was supported by the National Institutes of Health (grants no. 1DP2OD007394–01).
ON [18] G. P. Kotchey , B. L. Allen , H. Vedala , N. Yanamala , A. A. Kapralov ,
Y. Y. Tyurina , J. Klein-Seetharaman , V. E. Kagan , A. Star , ACS Nano 2011 , 5 , 2098 .
[19] International Agency for Research on Cancer , in IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans , Vol. 35 , 1985 .
[21] Chemical Summary: Hydrogen Peroxide, Environmental Protection Agency , http://actor.epa.gov/actor/GenericChemicalPdfServlet;jsessionid=D6A554DDB65663FCF3A4D193C182E719?casrn=7722/84/1 (accessed August 2013).
[22] M. Gulden , A. Jess , J. Kammann , E. Maser , H. Seibert , Free Radic. Biol. Med. 2010 , 49 , 1298 .
[23] a) H. Nakagawa , K. Hasumi , J. T. Woo , K. Nagai , M. Wachi , Carcino-genesis 2004 , 25 , 1567 ; b) D. R. Spitz , W. C. Dewey , G. C. Li , J. Cell. Physiol. 1987 , 131 , 364 .
[24] R. Heyrovska , arXiv:0804.4086 2008 . [25] M. S. Dresselhaus , A. Jorio , M. Hofmann , G. Dresselhaus , R. Saito ,
Nano Lett. 2010 , 10 , 751 . [26] J. Russier , C. Menard-Moyon , E. Venturelli , E. Gravel ,
G. Marcolongo , M. Meneghetti , E. Doris , A. Bianco , Nanoscale 2011 , 3 , 893 .
[27] R. P. Vidano , D. B. Fischbach , L. J. Willis , T. M Loehr , Solid State Commun. 1981 , 39 , 341 .
[28] A. C. Ferrari , J. C. Meyer , V. Scardaci , C. Casiraghi , M. Lazzeri , F. Mauri , S. Piscanec , D. Jiang , K. S. Novoselov , S. Roth , A. K. Geim , Phys. Rev. Lett. 2006 , 97 , 187401 .
[29] Z. Qu , G. Wang , J. Nanosci. Nanotechnol. 2012 , 12 , 105 . [30] J. Torreilles , M. C. Guerin , FEBS Lett. 1990 , 272 , 58 . [31] A. Reina , X. Jia , J. Ho , D. Nezich , H. Son , V. Bulovic ,
M. S. Dresselhaus , J. Kong , Nano Lett. 2009 , 9 , 30 . [32] a) C. Van Baalen , J. Marler , Nature 1966 , 211 , 951 ; b) D. Price ,
P. J. Worsfold , R. Fauzi , C. Mantoura , Anal. Chim. Acta 1994 , 298 , 121 .
