efremova_2014_toxicity of graphene shells, graphene oxide, and graphene

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ToxicityofGrapheneShellsGrapheneOxideandGrapheneOxidePaperEvaluatedwithEscherichiacoliBiotests

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MendeleevRussianUniversityofChemicalThellip


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DGDeryabin

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Research ArticleToxicity of Graphene Shells Graphene Oxide and GrapheneOxide Paper Evaluated with Escherichia coli Biotests

Ludmila V Efremova1 Alexey S Vasilchenko12

Eduard G Rakov3 and Dmitry G Deryabin1

1Department of Microbiology Orenburg State University Pobedy Avenue 13 Orenburg 460018 Russia2Institute of Cellular and Intracellular Symbiosis RAS Pionerskaya Street 11 Orenburg 460000 Russia3D Mendeleyev University of Chemical Technology Miusskaya Square 9 Moscow 125047 Russia

Correspondence should be addressed to Dmitry G Deryabin dgderyabinyandexru

Received 26 June 2014 Revised 29 September 2014 Accepted 3 October 2014

Academic Editor Lisa A Delouise

Copyright copy Ludmila V Efremova et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The plate-like graphene shells (GS) produced by an original methane pyrolysis method and their derivatives graphene oxide (GO)and graphene oxide paper (GO-P) were evaluated with luminescent Escherichia coli biotests and additional bacterial-based assayswhich together revealed the graphene-family nanomaterialsrsquo toxicity and bioactivity mechanisms Bioluminescence inhibitionassay fluorescent two-component staining to evaluate cell membrane permeability and atomic force microscopy data showed GOexpressed bioactivity in aqueous suspension whereas GS suspensions and the GO-P surface were assessed as nontoxic materialsThemechanismof toxicity ofGOwas shownnot to be associatedwith oxidative stress in the targeted soxSlux and katGlux reportercells also GO did not lead to significant mechanical disruption of treated bacteria with the release of intracellular DNA contentsinto the environment The well-coordinated time- and dose-dependent surface charge neutralization and transport and energeticdisorders in the Escherichia coli cells suggest direct membrane interaction internalization and perturbation (ie ldquomembranestressrdquo) as a clue to graphene oxidersquos mechanism of toxicity

1 Introduction

Graphene is a two-dimensional one-atom thick layer of car-bon packed into a honeycomb-like structure [1 2] Extremelythin carbon foils were predicted over more than 50 years ago[3] and the term of ldquographenerdquo was first introduced in 1987[4] but true graphene was only created by Novoselov et al in2004 [5]

Since then the technology of graphene and its derivativeshave been developing actively [6] The unique physicalproperties of graphene such as its exceptional mechanicalstrength thermal stability and high electrical conductivityattract attention in various fields of science and technology[7ndash9] The growing interest in graphene-family nanomate-rials (GFNs) is driving the study of their biological activityas well It is necessary to evaluate environmental risks ofgraphene-containing technological objects to biological sys-tems [10] as it is for other carbon-based nanomaterials [11]

in particular fullerenes [12] and nanotubes [13] Increasinginformation about graphene toxicity shows that its numberof layers lateral size stiffness hydrophobicity surface func-tionalization and dose are important [1 14ndash17] However thetoxicity and biocompatibility of GFNs are still debated [18]

Evaluating graphenersquos activity against bacteria is animportant step to understanding GFNsrsquo bioactivity Thesemodel organisms are responsive and sensitive to various dam-aging factors and their physiological manifestations allowunderstanding of toxicity mechanisms In 2010 Akhavan andGhaderi [19] first described the toxic effect of GFNs againstseveral bacterial species and also showed that grapheneoxide was more active compared to a pristine graphenesample Since then the toxicity of GFNs against bacteriahas been studied extensively (eg an ISI Web of Knowledgetopic search on 25062014 gave 168 hits for ldquographenerdquoand ldquobacteriardquo) but the results in dozens of publicationsare contradictory In particular this applies to so-called

Hindawi Publishing CorporationBioMed Research InternationalArticle ID 869361

2 BioMed Research International

(a) (b)

Figure 1 High resolution scanning electron microscopy image of the graphene shells (a) and atomic force microscopy image of grapheneoxide particles (b) Scale bar 500 nm

120579

120579 = 909∘

(a)

120579

120579 = 286∘

(b)

Figure 2 Examples of typical contact angles of the graphene-family nanomaterials surface (a) graphene shells and (b) graphene oxide

ldquographene paperrdquo [20] which showed an absence of effectsin some studies [21] while in other cases strong antibacterialactivity was reported [22 23]

Escherichia coli biotests are very attractive for solving thisproblem These bacteria are a wide-spread model organismin modern toxicology because well characterized physiologyand ease of genetic manipulation [24] In particular thebioassays based on recombinant luminescent light-off andlight-on E coli strains gave the possibility of obtaining thedetailed information about the biological activity of thetested compounds in real time manner [25] Previously inour studies the bioluminescence inhibition test has beenused to assess the toxicity of wide range of carbon-basednanomaterials [26] In turn in a recent study by Jia et al[27] the inducible luminescent E coli strains have beenused to assess the toxicity mechanisms of various carbonnanomaterials including graphene nanosheets

In the continuation of this research the aim of thisstudy was to evaluate some graphene-family nanomaterialsusing the luminescent Escherichia coli biotests and additionalbacterial-based assays which together reveal the GFNs toxic-ity and bioactivity mechanisms

2 Experimental

21 Graphene-Family Nanomaterials The graphene-familynanomaterials used in this work included graphene shells(GS) graphene oxide (GO) and graphene oxide paper(GO-P)

GS were synthesized through methane pyrolysis at 800∘Con MgO plates with hexagonal habitus and approximate sizeof hexagon edge 700 nm and average thickness of 115 nm[28] After MgO dissolution in diluted hydrochloric acid thehollow hexagonal aggregates 95 nm in thickness consisting ofseparate particles which contain several graphene layers wereobtained (Figure 1(a)) The specific surface area of the syn-thesizedmaterial was 1585m2g corresponding to a bilayer ofgraphene particlesGOwere prepared byGS anodic oxidationin sulfuric acid and subsequent oxidation with a mixtureof sulfuric and nitric acids under heating and microwaveirradiation [29] Atomic forcemicroscopy of the GO particles(Figure 1(b)) showed significant fragmentation of the plate-like shells as an indirect result of the oxidation process

GFNs covering solid surfaces were used for quantitativemeasurement of wetting by a polar liquid and work ofadhesion (119882

119886) calculations Briefly a 2 120583L drop of deionized

water was placed on the surface of a preformed GFN at20 plusmn 1

∘C and images were obtained instantaneously using adigital camera (Figure 2) From the images the values of thestatic contact angles were determined and 119882

119886values were

calculated by the Young-Dupre equation as 119882119886= 120590 times (1 +

cos 120579) where 120590 is the surface tension of water at 20∘C takenas 7286 times 0001Nm and 120579 is the average value of the staticcontact angle This integral parameter characterizing GFNsrsquohydrophobicityhydrophilicity was 744 plusmn 15 Nm and 1371 plusmn23Nm for GS and GO respectively

Aqueous suspensions of GS and GO (from 10minus3 to2 times 10

minus5mgL) were prepared in deionized water in vials

BioMed Research International 3

Table 1 Escherichia coli strains used in this study

Host strain Plasmid description Type of light emission ReferenceE coli K12 TG1 lac1015840luxCDABE Constitutive [26]E coli K12 MG1655 soxS1015840luxCDABE Inducible responsive to oxidative stress [30]E coli K12 MG1655 katG1015840luxCDABE Inducible responsive to oxidative stress [30]E coli K12 TG1 mdash Nonluminescent Collection strain

10

09

08

07

06

05

04

03

02

01

0

100 1e + 4 1e + 6

GOGS

Size distribution (nm)

Scat

terin

g am

plitu

de

Figure 3 Dynamic light scattering profiles for aqueous grapheneshells (GS) and graphene oxide (GO) suspensions

vigorously mixed and sonicated in an ultrasonic bath (SapfirRussia) at 35 kHz and a specific power of 30W times dmminus3 for30min The suspensions were then incubated for about 2hours at 20∘C thus allowing the colloidal systems to reachequilibrium Dynamic light scattering measurements wereperformed using the Photocor Compact-Z (Russia) Themeasured autocorrelation functions were analyzed and theeffective hydrodynamic radius (RH)was calculated accordingto the Einstein-Stokes Relation using the software suppliedwith the instrument (Figure 3)

The GS aqueous suspension was polydisperse and con-sisted of three fractions about 70 were large aggregateswith RH values of 67 plusmn 30 120583m and only 10 were particleswith RH = 45 plusmn 27 nm In turn 66 of the aggregates inthe GO suspension were characterized by an RH of 80 plusmn37 nm whereas lt10 of the aggregates were large particlesThus the preliminary characterization of the GFNs showedthem to be nano- and microscale carbon compounds whereGS is composed of hydrophobic plate-like shells which arepoorly dispersible in polar solvents whereas GO consistsof hydrophilic fragmentized particles which form a finelydispersed aqueous suspension

A GO suspension was used for graphene oxide paperproduction The GO-P was made by spraying the grapheneoxide shell colloid on a white cellulose chlorine-free Sveto-Copy paper 80 gm2 followed by air dryingThismethod gavea uniformly coated well-wetted and nonwaterproof surfaceof GFN material

22 Preparation of Bacterial Strains and Cells The experi-ments involving bacterial cells for GFN bioactivity assess-ments were performed using three Escherichia coliK12-basedlux-biosensors (Table 1)

The first was the commercially available E coli K12 TG1strain containing a recombinant plasmid with the luxCDABEoperon of Photobacterium leiognathi cloned under the lacpromoter which shows strong constitutive light emissionunder standard cultivation conditions Inhibition of its bio-luminescence is likely due to the toxicity associated withreducing bacterial metabolite levels or cell death becauselight production requires active cell metabolism LyophilizedE coli K12 TG1 laclux cells purchased from ldquoImmunotechrdquo(Russia) were rehydrated with cold distilled water to aconcentration of 37 times 108 colony-forming units (CFU) per1mL exposed for 30min at 2ndash4∘C and then the temperaturewas raised up to 25∘C before use

Two E coli K12 MG1655-based strains carried recom-binant plasmids containing a transcriptional fusion of hostsoxS or katG promoters to a truncated Photorhabdus lumi-nescens the luxCDABE operon These inducible biolumi-nescent strains exhibited low basal light emission thatincreases significantly during an oxidative stress responseas a consequence of simultaneous transcription of the soxSor katG-gene and lux-genes fusions [30] The bacteria weregrown on LB-agar (Sigma-Aldrich USA) supplemented with100 120583gmL of ampicillin for 18ndash24 hours at 37∘C after whichthe cells were transferred to fresh LB-broth and harvestedat an optical density of 01 absorption units at 545 nmcorresponding to a concentration of 25 times 107 colony-formingunits (CFU) per 1mL

The nonluminescent E coli K12 TG1 strain was used as acontrol for fluorescence and some additional bioassays (seethe following)

23 Bioluminescent Assays Light-off (bioluminescence inhi-bition) and light-on (bioluminescence induction) assays wereused for GFN bioactivity evaluations

To assess the GFNsrsquo toxicity we used a previouslydescribed version of bioluminescent analysis for carbon-based nanomaterials [26] Briefly sonicated aqueous GS andGO suspensions were added to the wells of a ldquoMicrolite 2+rdquo


microplate with nontransparent side walls (Thermo USA)wherein they were further doubly diluted in sterile distilledwater from 1 1 to 1 1024 up to a final volume of 50120583L Tothe filled wells was then added 50 120583L of a previously preparedsuspension of constitutively luminescent E coli K12 TG1laclux cells The key feature of the present experiment wasa set of serial dilutions of the GFNs so we could investigatevarious ratios of nanoparticles to cellsWells filled with steriledistilled water and containing an appropriate amount ofbacterial biosensor were used as controls

To assess the graphene oxide paper we used an originalvariation that provided placement of an intact sterile disc(Himedia India) into the wells followed by the placementof GO-P discs as a second layer and then applying 50 120583Lof the constitutively luminescent bacterial suspension to thesurface Second layers of white and black paper were used asthe controls

Bioluminescence measurements were carried out usingan LM-01T microplate luminometer (Immunotech CzechRepublic) which dynamically registered the luminescenceintensity of the samples for 180min estimated in relative lightunits (RLU) The data were analyzed using KILIA graphingsoftware To quantify the bioluminescence inhibition index(119868) due to GFN toxicity we used the algorithm 119868 = RLU119888

0times

RLU119905119899RLU119888

119899times RLU119905

0 where 119888 and 119905 are the RLU values of

the control and test samples at the 0th and 119899thminute ofmea-surement Based on these indexes we calculated the EC50toxicological parameters that is theGFN concentrations thatcause 50 inhibition of constitutive bioluminescence

The bioluminescence induction assay was conducted asfollows E coli K12 MG1655 soxSlux or E coli K12 MG1655katGlux were washed from the cultivation media and thenadjusted with 085 NaCl in water to obtain a final opticaldensity at 545 nm of 01 a 50120583L aliquot of the culture wasthen added to each well filled with a GO suspension asdescribed above Solvent only anduntreated cells were used asthe negative control samples Paraquat (final concentrations125ndash000305mmol) and H

2O2(000938ndash000002) were

added to the positive control wells as standards for oxidativestress induction in soxSlux and katGlux respectively After15 30 or 60min 100120583L of fresh LB-broth was added toeach well and the plates were incubated at 30∘C in a LM-01T luminometer Bioluminescence was monitored every3min for at least 120min without shaking The results werepresented as induction coefficients defined as the relativelight units (RLU) of an induced sample divided by that of theuntreated control samples

24 Fluorescent Bacterial Staining A two-component fluo-rescent LiveDead Baclight kit L-13152 (Molecular ProbesUSA) was used to evaluate membrane integrity in non-luminescent E coli K12 TG1 cells treated with GFNsThis direct-count assay utilizes two nucleic acid stainingdyes green-fluorescent SYTO9 (excitationemissionmaxima480500 nm) which labels all bacteria and red fluores-cent propidium iodide PI (490635 nm) which penetratesdamaged membranes only causing a reduction in SYTO 9fluorescence when both dyes are present Thus bacteria with

intact membranes stain fluorescent green whereas cells withincreased passively permeable membranes stain fluorescentred

Suspensions containing log-phase E coli K12 TG1 cells(OD540

= 001) in distilled water were mixed with GS andGO suspensions placed on the GO-P surface and incubatedfor 60 120 and 180 minutes in a moist chamber Fordyeing a portion of a liquid sample or paper fragmentswere transferred to nonfluorescentmicroscopy glass and sub-jected to one-step staining according to the manufacturerrsquosrecommendation The bacteriarsquos colors were observed in aMikromed-3-Lum fluorescent microscope (Russia) equippedwith filter sets useful for simultaneous viewing of the SYTO9 and PI stains images for cell quantification were thenacquired with a digital camera

25 Atomic Force Microscopy Visualization of contactsbetween GFNs and bacterial cells was performed using anatomic force microscope SMM-2000 (Proton-MIET Russia)as described previously [31] Briefly aliquots (20120583L) of intactbacterial cells alone or mixed with GS or GO suspensionswere applied to freshly prepared mica and intact bacterialcells were applied to the GO-P surface The samples wereincubated at 93 relative humidity and 20ndash22∘C and scannedin a contact mode using V-shaped silicon nitride cantilevers(MSCT-AUNM Veeco Instruments Inc USA) with a springconstant of 001Nm and a tip curvature of 10 nm Quanti-tative morphometric analysis of the images was performedusing the standard software provided with the instrument

26 Efflux of Intracellular DNA Content The plasmid-containing E coliK12 TG1 laclux strain was cultured inmid-exponential growth phase with ampicillin and then separatedfrom its cultivation medium and incubated with aqueousGO suspensions at concentrations of 125 times 10minus4 5 times 10minus4and 2 times 10minus3mgL for 60min or with solvent only (negativecontrol) or 05 SDS solution which gives rapid 100 lysisof all bacterial cells (positive control) After centrifugation at14000 g for 15min (Eppendorf Germany) the concentrationof total DNA in the supernatant was analyzed by fluores-cence spectroscopy (Solar CM2203 Belarus) using ethidiumbromide as a fluorescent dye (excitationemission maxima285605 nm) Salmon sperm DNA was used as an externalstandard (0ndash3 120583gmL)

27 Zeta Potential Measurement The zeta potential (mem-brane surface charge) of E coli K12 TG1 cells was determinedusing Photocor Compact-Z (Russia) equipment A bacterialsuspension (37 times 108 CFUmL) was mixed with seriallydiluted GO aqueous suspensions to a final concentrationfrom 31 times 10minus5 to 5 times 10minus4mgL Measurements were carriedout in triplicate for two samples at 60 120 and 180 minutesZeta potentials were calculated from the electrophoretic cellmobility data obtained from Smoluchowskirsquos equation

28 Statistics The results were processed statistically by themethod of variance using the software package Statistica V8(StatSoft Inc United States)


10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)

Figure 4 The time course of E coli K12 TG1 laclux luminescence during contact with graphene shells (a) and graphene oxide (b)aqueous suspensions or graphene oxide paper and control paper (c) Ordinate-luminescence level RLU abscissa-time measurement minDesignations 1 10minus3mgL 2 5 times 10minus4mgL 3 25 times 10minus4mgL 4 125 times 10minus4mgL 5 625 times 10minus5mgL 6 313 times 10minus5mgL 7 156 times 10minus5mgL8 781 times 10minus6mgL 9 391 times 10minus6mgL C control wp white paper bp black paper GO-P graphene oxide paper

3 Results and Discussion

31 Comparative Evaluation of GFNs againstEscherichia coli Cells

311 GFN Toxicity Assessment Using a Bacterial Biolumi-nescence Inhibition Assay Measurement of E coli K12 TG1laclux luminescence in contact with GFNs revealed rapidlight inhibition in the first second of instrument registration(Figure 4) The GS suspension showed a less pronouncedeffect in contrast to the GO suspension which dose-dependently decreased the luminescence intensity In ourexperience [26] this ldquofalserdquo effect was determined by opticaldistortion in GFNs suspensions not being associated withcarbon-based nanomaterials bioactivity

A similar result was apparent during the contact betweenE coli K12 TG1 laclux and the paper samples The lumi-nescence intensity decreased significantly in the first secondwhen the biosensor was applied on GO-P or to a controlblack paper while the luminescence intensitywith the controlwhite paper was high due to this materialrsquos minimal lightabsorption and maximal light reflection

Continued luminescence measurement showed differentlight intensity changes due to biosensor contact with the GSsuspension and GO-P on the one hand and the GO suspen-sion on the other The poorly dispersible GS suspension wasevaluated as nontoxic because of the absence of significantluminescence inhibition (Figure 4(a)) In contrast the finelydispersed GO suspension led to inhibition of luminescenceand in some cases to zero luminescence depending on theconcentration and with increasing time (Figure 4(b)) Thiseffect was interpreted as ldquotruerdquo toxicity of graphene oxideagainst the E coli K12 TG1 laclux biosensor In the currentsystem containing 37 times 108 CFUmL cells GO toxicity wascharacterized by EC50 values of 113 plusmn 005 times 10minus4 104 plusmn

003 times 10minus4 and 792 plusmn 024 times 10minus5mgL after 60 120 and180min of measurement respectively In turn the initialdecrease in E coli K12 TG1 laclux luminescence on the GO-P surface accompanied by a very slow further decline in lightproduction (Figure 4(c)) led to the evaluation of thismaterialas nontoxic

Thus the bioluminescence inhibition assays showeda GFNrsquos bioactivity was dependent on its hydrophilic-itydispersivity (in the case of the GS versus GO comparison)and on the type of interaction with the bacterial biosensor(the GO suspension was toxic while the surface of the GO-Pwas not)

The second step in assessing graphene oxide toxicityagainst the E coli K12 TG1 laclux strain examined variousGO cell suspension ratios This approach allowed us toanalyze the relationships between the surface areas whichwere calculated based on bacterial CFU per 1mL and DLS-data respectively

The bioluminescence inhibition assay showed that GOtoxicity was dependent upon the content of bacterial cellsin the sample (Figure 5) whereby the EC50 values decreasedwith decreasing the CFU number (Table 2) At the 60th minof measurement these values varied from 113 plusmn 005 times 10minus4to 255 plusmn 010 times 10minus5mgL following twofold bacterial celldilutions These data showed the need to cover the bacterialsurface with a certain number of GO particles in order forthe toxic effect to be apparent So the calculation of GO cellratios gave a stable value of 05 plusmn 005 120583m2 GO surface for1 120583m2 bacterial surface as necessary for 50 bioluminescenceinhibition Prolonging the bioluminescence measurementto 120 and 180min revealed an ongoing toxic effect thatis evident in Table 2 by the decreasing EC50 values Thisobservation shows the dose-dependency as well as time-dependency of GO toxicity in aqueous suspension


Table 2 Dose-dependent and time-dependent GO aqueous suspensions toxicity in bacterial bioluminescence inhibition assay characterizedby EC50 value (mgL content) at varying bacterial cell number in the sample

E coli K12 TG1 laclux CFU per 1mL Time measurement60min 120min 180min

370 times 108 113 plusmn 005 times 10minus4 104 plusmn 003 times 10minus4 792 plusmn 024 times 10minus5




15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I

Figure 5 The dose-response curves described the changes in thebioluminescence inhibition indexes (119868) caused by differing grapheneoxide (GO) content and varying bacterial cell number in the sample1ndash370 times 108 CFU per 1mL 2ndash185 times 108 CFU per 1mL 3ndash925 times107 CFU per 1mL 4ndash462 times 107 CFU per 1mL

312 GFN Bioactivity in a Fluorescent Cell Assay Two-component differential fluorescent labeling of the nonlu-minescent E coli K12 TG1 strain detected 999 plusmn 01green-fluorescent cells in both samples treated with GS andGO-P (Figures 6(a) and 6(c)) indicating intact membranesconsistent with the bioluminescent assay results In contrastbacterial cells treated with GO suspensions became red-fluorescent after 60min in a dose-dependent manner from488 plusmn 19 (at 125 times 10minus4mgL GO) to 930 plusmn 28 (at 5 times10minus4mgL GO) because of membrane damage that allowedthe fluorescent PI dye to penetrate (Figure 6(b)) Prolongingthe exposure to 180min increased the red-fluorescent cellfraction to 739 plusmn 22ndash947 plusmn 33 These data suggestedGO bioactivity against the E coli cells and showed increasedpassive membrane permeability and bioluminescence inhibi-tion as simultaneous toxicity indicators The data from thebioluminescent and fluorescent assays were nonidentical butwell correlated as will be discussed below (see Section 323)

313 AFM-Evaluation of GFN and Escherichia coli CellInteractions E coli K12 TG1 cells treated with GS and GOsuspensions or applied on the surface of GO-P were trans-ferred to mica and visualized by AFM-microscopy (Figures6(d)ndash6(f))

This study did not reveal any differences in GS-treatedcells in shape and size (122 120583m3 in volume) compared tocontrol intact bacteria Cell contact with GS was also notdetected (Figure 6(d)) despite large 05ndash30 120583maggregates onthemica surface around the cells being visualized In contrast

E coli K12 TG1 cells incubated with GO suspensions hadsignificantly altered morphology (Figure 6(e)) The bacterialcell surface was covered with numerous 67 plusmn 13 nm particlesthat were apparent around the cells too and correspondedwell to the GO particle sizes revealed by dynamic lightscattering (see Section 21) As a result the roughness ofthe GO-covered bacterial surface was more than twofoldgreater in comparison with the control cells (119875 lt 0001)The volume (081 120583m3 119875 lt 001) of GO-treated cells alsodiffered from those of control cells Moreover some GO-treated cells changed their morphology more significantlyand were visualized spread-eagled on the mica surfacecharacteristic of membrane permeability damage At thesame timeE coliK12TG1 cells lying on the surface of theGO-Pwere indistinguishable from the control samples in terms ofvolume (112 120583m3) as well as not showing any AFM-detectedlesions (Figure 6(f))

Thus three assays including bioluminescence fluores-cence and AFM showed the toxicity of a GO suspensionagainst Escherichia coli cells and established the absenceof toxicity of a GS suspension or GO-covered paper Theobtained results were the basis for further research into themechanisms of graphene oxide toxicity

32 Evaluation of GO Toxicity Mechanisms againstEscherichia coli Cells

321 GO Validation as a Potential Oxidative Stress Inducerin Lux-Biotests The E coli K12 MG1655 soxSlux and E coliK12MG1655 katGlux strains were used in a bioluminescenceinduction assay for oxidative stress as reflected in increasedlight emission

The luminescent response of these strains can be veri-fied with model oxidants such as paraquat - 111015840-dimethyl-441015840-bipyridinium dichloride (Sigma USA) causing electrontransfer from bacterial respiratory chains onto molecularoxygen with intracellular superoxide-anion production or inthe presence of exogenous hydrogen peroxide (Figure 7) Ecoli K12 MG1655 soxSlux cells treated with paraquat con-centrations as low as 000305mmol exhibited much strongerdose-dependent luminescence as a consequence of simul-taneous transcription of the soxS-gene and soxSlux genesfusions A peak 87-fold increase in bioluminescence wasdetected with 004883mmol paraquat treatment whereas thehighest concentrations of the inducer resulted in inhibitionof luminescence because of their extreme toxic effect Inturn E coliK12MG1655 katGluxwas activated by hydrogen


(a) (b) (c)

(d) (e) (f)

Figure 6 Fluorescentmicroscopy images ((a)ndash(c)) and atomic forcemicroscopy images ((d)ndash(f)) ofE coliK12TG1 cells treatedwith grapheneshells suspension at 10minus3mgL for 180min ((a) (d)) and graphene oxide suspension at 125 times 10minus4mgL for 180min ((b) (e)) and placed onthe surface of graphene oxide paper for 60min ((c) (f)) Scale bar 5 120583m ((a)ndash(c)) 1 120583m ((d)ndash(f))

100

80

60

40

20

0

0

10E minus 4 20E minus 4 30E minus 4 40E minus 4 50E minus 4

I

rfl

z

GO (mgL)

()

Figure 7The relationship between E coliK12 TG1 cellsrsquo zeta poten-tial values (119911) red fluorescent labeling (rfl) and bioluminescenceinhibition indices (119868) depended on the graphene oxide (GO) contentin the suspensions

peroxide at a concentration of 000002 and achieved amaximal 176-fold induction at 000469

Bacterial luminescence measurement in the presenceof the GO suspension did not show significant inductioncoefficients The 12ndash15-fold light intensity growth onlyhas been recorded at subtoxic GO concentrations (195 times10minus6ndash10minus3mgL) In the current experimental context it wasunlikely the stress response more likely it was a recoveryof bioluminescent reaction after nutrient media supplemen-tation than stress response In turn GO concentrations of

2 times 10minus3mgL or more were toxic and resulted in irreversiblebacterial luminescence inhibition in the E coli K12 MG1655soxSlux and E coli K12 MG1655 katGlux strains just aswas shown in the E coli K12 TG1 laclux luminescence assay(see Section 311) Thus the mechanism of GO toxicity is notassociated with oxidative stress in the targeted E coli cells

322 Intracellular DNA Efflux Measurement in GO-TreatedEscherichia coli Cells The toxicity caused by cellular mem-brane damage was further verified bymeasuring intracellularDNA efflux by plasmid-containing E coli K12 TG1 cellsexposed to GO The DNA concentration in intact controlsupernatants was undetectable using a fluorescence spec-troscopy assay with ethidium bromide as a fluorescent dye Incontrast DNA was easy to detect in the supernatant of E colitreated with SDS and was quantified as 3 120583gmL according toDNA standards In turn DNA levels in supernatants of cellsexposed to GO at concentrations of 125 times 10minus4ndash2 times 10minus3mgLfor 60ndash180min were similar to those of the intact controlwhereas both the luminescent and fluorescent assays showeda toxic effect Thus we report that GO-induced membranedamage did not lead to cell lysis reflected in an efflux ofbiopolymers (including plasmid DNA) but their increasedpassive permeability was rather limited to small molecules(including fluorescent dyes)


323 Effect of GO Treatment on Zeta Potential Propertiesof Escherichia coli Cells Zeta potential studies were carriedout to reveal the effect of GO on the membrane surfacecharge in E coli K12 TG1 cells The control bacterial sampledisplayed a zeta potential value of minus3131 plusmn 094mV whichgradually decreased to minus2419 plusmn 072mV during dynamicmeasurement Upon the addition of increasing GO concen-trations the E coli zeta potential values decreased slightlyupon initial contact and then the membrane surface chargewas progressively neutralized in a dose-dependent mannerfrom 60min (Figure 7) until 180min Finally the GO con-centration required for complete zeta potential neutralizationwas 250 times 10minus4mgL whereas concentrations in the range of313 times 10minus5ndash125 times 10minus4mgL were sufficient to promote zetapotential values from minus1849 plusmn 149mV to minus944 plusmn 002mVThese data show that zeta potential neutralization is animportant result of GO nanoparticlesrsquo interaction with bac-terial surfaces having the negative charge originating fromouter lipopolysaccharide molecules and using this electricalpotential in a membrane-associated energetic process

Interestingly measurements of E coli cells treated withincreasing GO concentrations established a correlationbetween zeta potential neutralization and bioluminescenceinhibition values at 60min (119903 = 0973 119875 lt 001) Thiscan be interpreted as GO targeting of electrical potentialacross cell membranes that affected the energy metabolismavailable for biochemical reactions in a bacterial cell includ-ing those required for energetic bioluminescent substrateswherein the bacterial bioluminescence inhibition assay wasthe most sensitive which allows us to recommend it toassess a GFNrsquos toxicity The two-component fluorescent dataevaluating cell membrane permeability and zeta potentialmeasurement also correspond well (119903 = 0943 119875 lt 001)indicating a dual violation of bacterial membrane functionsThe similar trends for these parameters (Figure 7) suggestmembrane perturbation (ie ldquomembrane stressrdquo) is a clueto the mechanism of grapheme oxide toxicity in which GOadhesion and internalization processes trigger transport andenergetic disorders

4 Conclusion

The toxicity of graphene-family nanomaterials includingpristine grapheme against bacteria was first described in2010 [19] but subsequent results have been contradictory andunderstanding GFNsrsquo mechanisms of toxicity is incomplete[18 32] In this work we studied graphene shells synthesizedusing an original methane pyrolysis method [28] a grapheneoxide derivative [29] and graphene oxide paper in severalEscherichia coli biotests sufficient to reveal the GFNsrsquo toxicityand bioactivity mechanisms

Combining a bioluminescence inhibition assay and two-component fluorescence in order to evaluate cell membranepermeability with atomic force microscopy there was anabsence of detectable bioactivity for GS Although the GSsample used differed from canonical graphene and containedseveral (usually two) layers the data clearly show this type ofGFN is nontoxic We attribute this result to GS not being well

dispersed in water or other polar solvents [33] whereas thiscondition is a prerequisite for bioassays as in real biologicalsystems

The oxidation reaction led to graphene fragmentation aswell as increased hydrophilicity and the highly dispersibleGOparticles interacted stronglywith the bacterial cell surfaceand caused a time- and dose-dependent toxic effect Thuswe confirm the high toxicity of graphene oxide againstbacterial cells compared to its nonreduced counterpart [1419] determined by significant changes in its physicochem-ical properties The revealed GO toxicity mechanism canbe summarized as follows (i) substantial covering of thebacterial surface by GO is a necessary prerequisite for themanifestation of bioactivity (ii)GOadhesion causes bacterialsurface charge neutralization (iii) GO internalization bythe cell membrane leads to increased passive membranepermeability including PI dye (iv) energetic processes inGO-treated cells were violated resulting in bioluminescenceinhibition as the most sensitive parameter for carbon-basednanomaterial toxicity evaluation In contrast we did notfind significant mechanical disruption of GO-treated cellswith the release of intracellular DNA into the environmentor oxidative stress in the targeted bacterial cells [34 35]indicating ldquomembrane stressrdquo as a clue to the mechanism ofgraphene oxide toxicity

The GO-covered paper surface was nontoxic forEscherichia coli cells in agreement with [21 36] but incontradiction to [22 23] which showed high antibacterialactivity by graphene paper In our opinion the biologicalinertness of GO-P was determined by its targeting only one-half of the bacterial surface whereas the intact membraneprovided a sufficient surface for vital functions Moreoverin contrast to pristine graphene graphene oxide is notelectrically conductive while charge transfer is thought to bea leading cause of the antibacterial property of graphene film[37] Finally the absence of impurities or metal nanoparticles[38 39] also resulted in the nontoxicity of the used GO-Psample

In summary our results showed the importance ofthe physicochemical properties of graphene-family nano-materials as well as the quality of their suspensionsor spatial surface organization for their toxicity againstEscherichia coli cells We revealed the expressed grapheneoxide bioactivity in aqueous suspensions whereas grapheneshells and graphene oxide paper were determined to benontoxic materials The obtained result requires the reg-ulation of GOrsquos presence in the environment [24 40]as well as confirming that the antibacterial property ofGO has the potential to be useful [19 38] In pursu-ing biomedical applications great care must be taken toensure the toxicity or safety of each GFN against bacte-rial and eukaryotic cells is well characterized and under-stood

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors thank the Russian Ministry of Education andScience (project no 148) Orenburg Region Government(project no 20) and the Russian Foundation for BasicResearch (project no 15-04-04379 A) for financial support

References

[1] H Zhang G Gruner and Y Zhao ldquoRecent advancements ofgraphene in biomedicinerdquo Journal ofMaterials Chemistry B vol1 no 20 pp 2542ndash2567 2013

[2] A K Geim and K S Novoselov ldquoThe rise of graphenerdquo NatureMaterials vol 6 no 3 pp 183ndash191 2007

[3] H P Boehm A Clauss G O Fischer and U Hofmann ldquoDasAdsorptionsverhalten sehr dunnerKohlenstoffolienrdquoZeitschriftfur Anorganische und Allgemeine Chemie vol 316 no 3-4 pp119ndash127 1962 (German)

[4] A Hamwi S Mouras D Djurado and J C Cousseins ldquoNewsynthesis of first stage graphite intercalation compounds withfluoridesrdquo Journal of Fluorine Chemistry vol 35 no 1 p 1511987

[5] K S Novoselov A K Geim S V Morozov et al ldquoElectric fieldin atomically thin carbon filmsrdquo Science vol 306 no 5696 pp666ndash669 2004

[6] K S Novoselov ldquoTechnology rapid progress in producinggraphenerdquo Nature vol 505 no 7483 p 291 2014

[7] C N R Rao A K Sood K S Subrahmanyam and AGovindaraj ldquoGraphene the new two-dimensional nanomate-rialrdquo Angewandte Chemie International Edition vol 48 no 42pp 7752ndash7777 2009

[8] S Guo and SDong ldquoGraphene nanosheet Synthesismolecularengineering thin film hybrids and energy and analyticalapplicationsrdquoChemical Society Reviews vol 40 no 5 pp 2644ndash2672 2011

[9] H Jang J Lee and D-H Min ldquoGraphene oxide forfluorescence-mediated enzymatic activity assaysrdquo Journalof Materials Chemistry B vol 2 no 17 pp 2452ndash2460 2014

[10] D Bradley ldquoIs graphene saferdquoMaterials Today vol 15 no 6 p230 2012

[11] A C L Tang G-L Hwang S-J Tsai et al ldquoBiosafety of non-surface modified carbon nanocapsules as a potential alternativeto carbon nanotubes for drug delivery purposesrdquo PLoS ONEvol 7 no 3 Article ID e32893 2012

[12] T Mashino D Nishikawa K Takahashi et al ldquoAntibacterialand antiproliferative activity of cationic fullerene derivativesrdquoBioorganic amp Medicinal Chemistry Letters vol 13 no 24 pp4395ndash4397 2003

[13] G Jia HWang L Yan et al ldquoCytotoxicity of carbon nanomate-rials single-wall nanotube multi-wall nanotube and fullerenerdquoEnvironmental Science and Technology vol 39 no 5 pp 1378ndash1383 2005

[14] S Liu T H Zeng M Hofmann et al ldquoAntibacterial activity ofgraphite graphite oxide graphene oxide and reduced grapheneoxide membrane and oxidative stressrdquo ACS Nano vol 5 no 9pp 6971ndash6980 2011

[15] C M Santos M C R Tria R A M V Vergara F AhmedR C Advincula and D F Rodrigues ldquoAntimicrobial graphenepolymer (PVK-GO) nanocomposite filmsrdquo Chemical Commu-nications vol 47 no 31 pp 8892ndash8894 2011

[16] A Schinwald F A Murphy A Jones W MacNee and KDonaldson ldquoGraphene-based nanoplatelets a new risk to therespiratory system as a consequence of their unusual aerody-namic propertiesrdquo ACS Nano vol 6 no 1 pp 736ndash746 2012

[17] V C Sanchez A Jachak R H Hurt and A B Kane ldquoBiologicalinteractions of graphene-family nanomaterials an interdisci-plinary reviewrdquo Chemical Research in Toxicology vol 25 no 1pp 15ndash34 2012

[18] A Bianco ldquoGraphene safe or toxic the two faces of the medalrdquoAngewandte Chemie International Edition vol 52 no 19 pp4986ndash4997 2013

[19] O Akhavan and E Ghaderi ldquoToxicity of graphene andgraphene oxide nanowalls against bacteriardquo ACS Nano vol 4no 10 pp 5731ndash5736 2010

[20] D A Dikin S Stankovich E J Zimney et al ldquoPreparation andcharacterization of graphene oxide paperrdquo Nature vol 448 no7152 pp 457ndash460 2007

[21] S ParkNMohanty JW Suk et al ldquoBiocompatible robust free-standing paper composed of a TWEENgraphene compositerdquoAdvanced Materials vol 22 no 15 pp 1736ndash1740 2010

[22] W Hu C Peng W Luo et al ldquoGraphene-based antibacterialpaperrdquo ACS Nano vol 4 no 7 pp 4317ndash4323 2010

[23] L Yu Y Zhang B Zhang J Liu H Zhang and C SongldquoPreparation and characterization of HPEI-GOPES ultrafiltra-tion membrane with antifouling and antibacterial propertiesrdquoJournal of Membrane Science vol 447 pp 452ndash462 2013

[24] S Girotti E N FerriM G Fumo and EMaiolini ldquoMonitoringof environmental pollutants by bioluminescent bacteriardquo Ana-lytica Chimica Acta vol 608 no 1 pp 2ndash29 2008

[25] A Ivask O Bondarenko N Jepihhina and A Kahru ldquoProfilingof the reactive oxygen species-related ecotoxicity of CuO ZnOTiO2

silver and fullerene nanoparticles using a set of recom-binant luminescent Escherichia coli strains differentiating theimpact of particles and solubilised metalsrdquo Analytical andBioanalytical Chemistry vol 398 no 2 pp 701ndash716 2010

[26] D G Deryabin E S Aleshina and L V Efremova ldquoApplicationof the inhibition of bacterial bioluminescence test for assess-ment of toxicity of carbon-based nanomaterialsrdquoMicrobiologyvol 81 no 4 pp 492ndash497 2012

[27] K Jia R SMarks andR E Ionescu ldquoInfluence of carbon-basednanomaterials on lux-bioreporter Escherichia colirdquo Talanta vol126 pp 208ndash213 2014

[28] S Y Davydov A Y Kryukov V O Gerya I M Izvolrsquoskii and EG Rakov ldquoPreparation of a platelike carbon nanomaterial usingMgO as a templaterdquo Inorganic Materials vol 48 no 3 pp 244ndash248 2012

[29] H V Nguyen N M Tun A Y Kryukov I M Izvolrsquoskii and EG Rakov ldquoDependence of the ldquosolubilityrdquo of oxidized carbonnanomaterials on the acidity of aqueous solutionsrdquo RussianJournal of Physical Chemistry A vol 88 no 9 pp 1559ndash15632014

[30] V Y Kotova I V Manukhov and G B Zavilgelskii ldquoLux-biosensors for detection of SOS-response heat shock andoxidative stressrdquoApplied Biochemistry andMicrobiology vol 46no 8 pp 781ndash788 2010

[31] H Nikiyan A Vasilchenko and D Deryabin ldquoHumidity-dependent bacterial cells functional morphometry investiga-tions using atomic forcemicroscoperdquo International Journal ofMicrobiology vol 2010 Article ID 704170 5 pages 2010

[32] A M Jastrzebska P Kurtycz and A R Olszyna ldquoRecentadvances in graphene family materials toxicity investigationsrdquoJournal of Nanoparticle Research vol 14 no 12 p 1320 2012


[33] D Li M B Muller S Gilje R B Kaner and G G WallaceldquoProcessable aqueous dispersions of graphene nanosheetsrdquoNature Nanotechnology vol 3 no 2 pp 101ndash105 2008

[34] S M Notley R J Crawford and E P Ivanova ldquoBacterialinteraction with graphene particles and surfacesrdquo in Advancesin Graphene Science M Aliofkhazraei Ed 2013

[35] Y Zhang S F Ali E Dervishi et al ldquoCytotoxicity effectsof graphene and single-wall carbon nanotubes in neuralphaeochromocytoma-derived pc12 cellsrdquo ACS Nano vol 4 no6 pp 3181ndash3186 2010

[36] H Chen M B Muller K J Gilmore G G Wallace and D LildquoMechanically strong electrically conductive and biocompat-ible graphene paperrdquo Advanced Materials vol 20 no 18 pp3557ndash3561 2008

[37] J Li G Wang H Zhu et al ldquoAntibacterial activity of large-area monolayer graphene film manipulated by charge transferrdquoScientific Reports vol 4 article 4359 2014

[38] B-S Wu H N Abdelhamid and H-F Wu ldquoSynthesis andantibacterial activities of graphene decorated with stannousdioxiderdquo RSC Advances vol 4 no 8 pp 3722ndash3731 2014

[39] Y-W Wang A Cao Y Jiang et al ldquoSuperior antibacterialactivity of zinc oxidegraphene oxide composites originatingfrom high zinc concentration localized around bacteriardquo ACSApplied Materials and Interfaces vol 6 no 4 pp 2791ndash27982014

[40] C Pretti M Oliva R D Pietro et al ldquoEcotoxicity of pristinegraphene tomarine organismsrdquo Ecotoxicology and Environmen-tal Safety vol 101 no 1 pp 138ndash145 2014

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of

Research ArticleToxicity of Graphene Shells Graphene Oxide and GrapheneOxide Paper Evaluated with Escherichia coli Biotests

Ludmila V Efremova1 Alexey S Vasilchenko12

Eduard G Rakov3 and Dmitry G Deryabin1

1Department of Microbiology Orenburg State University Pobedy Avenue 13 Orenburg 460018 Russia2Institute of Cellular and Intracellular Symbiosis RAS Pionerskaya Street 11 Orenburg 460000 Russia3D Mendeleyev University of Chemical Technology Miusskaya Square 9 Moscow 125047 Russia

Correspondence should be addressed to Dmitry G Deryabin dgderyabinyandexru

Received 26 June 2014 Revised 29 September 2014 Accepted 3 October 2014

Academic Editor Lisa A Delouise

Copyright copy Ludmila V Efremova et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The plate-like graphene shells (GS) produced by an original methane pyrolysis method and their derivatives graphene oxide (GO)and graphene oxide paper (GO-P) were evaluated with luminescent Escherichia coli biotests and additional bacterial-based assayswhich together revealed the graphene-family nanomaterialsrsquo toxicity and bioactivity mechanisms Bioluminescence inhibitionassay fluorescent two-component staining to evaluate cell membrane permeability and atomic force microscopy data showed GOexpressed bioactivity in aqueous suspension whereas GS suspensions and the GO-P surface were assessed as nontoxic materialsThemechanismof toxicity ofGOwas shownnot to be associatedwith oxidative stress in the targeted soxSlux and katGlux reportercells also GO did not lead to significant mechanical disruption of treated bacteria with the release of intracellular DNA contentsinto the environment The well-coordinated time- and dose-dependent surface charge neutralization and transport and energeticdisorders in the Escherichia coli cells suggest direct membrane interaction internalization and perturbation (ie ldquomembranestressrdquo) as a clue to graphene oxidersquos mechanism of toxicity

1 Introduction

Graphene is a two-dimensional one-atom thick layer of car-bon packed into a honeycomb-like structure [1 2] Extremelythin carbon foils were predicted over more than 50 years ago[3] and the term of ldquographenerdquo was first introduced in 1987[4] but true graphene was only created by Novoselov et al in2004 [5]

Since then the technology of graphene and its derivativeshave been developing actively [6] The unique physicalproperties of graphene such as its exceptional mechanicalstrength thermal stability and high electrical conductivityattract attention in various fields of science and technology[7ndash9] The growing interest in graphene-family nanomate-rials (GFNs) is driving the study of their biological activityas well It is necessary to evaluate environmental risks ofgraphene-containing technological objects to biological sys-tems [10] as it is for other carbon-based nanomaterials [11]

in particular fullerenes [12] and nanotubes [13] Increasinginformation about graphene toxicity shows that its numberof layers lateral size stiffness hydrophobicity surface func-tionalization and dose are important [1 14ndash17] However thetoxicity and biocompatibility of GFNs are still debated [18]

Evaluating graphenersquos activity against bacteria is animportant step to understanding GFNsrsquo bioactivity Thesemodel organisms are responsive and sensitive to various dam-aging factors and their physiological manifestations allowunderstanding of toxicity mechanisms In 2010 Akhavan andGhaderi [19] first described the toxic effect of GFNs againstseveral bacterial species and also showed that grapheneoxide was more active compared to a pristine graphenesample Since then the toxicity of GFNs against bacteriahas been studied extensively (eg an ISI Web of Knowledgetopic search on 25062014 gave 168 hits for ldquographenerdquoand ldquobacteriardquo) but the results in dozens of publicationsare contradictory In particular this applies to so-called

Hindawi Publishing CorporationBioMed Research InternationalArticle ID 869361


(a) (b)


120579

120579 = 909∘

(a)

120579

120579 = 286∘

(b)





2 Experimental







119886values were








10

09

08

07

06

05

04

03

02

01

0

100 1e + 4 1e + 6

GOGS


Scat

terin

g am

plitu

de















0times

RLU119905119899RLU119888

119899times RLU119905




2O2(000938ndash000002) were











10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)


















15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)


120579

120579 = 909∘

(a)

120579

120579 = 286∘

(b)





2 Experimental







119886values were








10

09

08

07

06

05

04

03

02

01

0

100 1e + 4 1e + 6

GOGS


Scat

terin

g am

plitu

de















0times

RLU119905119899RLU119888

119899times RLU119905




2O2(000938ndash000002) were











10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)


















15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




10

09

08

07

06

05

04

03

02

01

0

100 1e + 4 1e + 6

GOGS


Scat

terin

g am

plitu

de















0times

RLU119905119899RLU119888

119899times RLU119905




2O2(000938ndash000002) were











10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)


















15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





0times

RLU119905119899RLU119888

119899times RLU119905




2O2(000938ndash000002) were











10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)


















15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


10000

1000

100

10

1

1

0 50 100 150 200

(min)

C

RLU

2ndash9

(a)

10000

1000

100

10

1

7

6

5

4321

0 50 100 150 200

(min)

C

RLU

8-9

432111

(b)

wp

bp

GO-P

10000

1000

100

10

1

0 50 100 150 200

(min)

RLU

(c)


















15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








15

1

05

0

4 3 2 1

0

25Eminus5

50Eminus5

75Eminus5

10Eminus4

125Eminus4

15Eminus4

175Eminus4

20Eminus4

225Eminus4

250Eminus4

GO (mgL)

I











(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b) (c)

(d) (e) (f)


100

80

60

40

20

0

0


I

rfl

z

GO (mgL)

()









4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




4 Conclusion










Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of














Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

efremova_2014_toxicity of graphene shells, graphene oxide, and graphene

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