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found to be exceptionally biocompatible against MDCK cells. 2013 Elsevier B.V. All rights reserved.

al uouoresdue tome inideal c

applications are magnicent uorescent pro

broad excitation spectra, and narrow and tunable emission spectra C-dots we are reporting synthesis of highly luminescent bio-friendly

Materials Science and Engineering C 33 (2013) 29142917

Contents lists available at SciVerse ScienceDirect

Materials Science a

.e l[1,3,4]. However, involvement of cadmium and arsenic in the synthe-sis of QDsmakes thempotentially hostile towards the biological appli-cations [4,5].

With the advancement in carbon nanomaterials such as carbonnanotubes [6], fullerenes [7] have become common ingredients ofmaterial engineering. Carbon dots are emerging novel materials withdesirable uorescent properties and have been used in bio-imaging[8]. There aremany established protocols for the synthesis of lumines-cent C-dots such as microwave mediated synthesis [2], laser ablationof graphite [9,10], thermal cracking of organic compounds [11,12],

C-dots coatedwith natural linkers tomake them stable in the solution.It can also be used as linkers for the attachment of the drugs fortargeted drug delivery. This is the rst report of the use of plant mate-rials for the synthesis of C-dots without using any external oxidizingagent such as ethanol used in previous reports mentioned above.The absence of any noxious chemical makes our process completelysafe for any biological application. To verify this, we have given cyto-toxicity effect of C-dots on MDCK cells. Results show remarkable bio-compatibility at very high concentrations of C-dots.electro-oxidation of graphite [13], and oxidaFluorescent properties of the dots can be tumodications such as C-dots functionalized whibits blue orescence [15] while those orc

Corresponding authors. Tel.: +91 9004024937, +9E-mail addresses: gurus.spandey@gmail.com (S. Pan

sharonmadhuri@gmail.com (M. Sharon).

0928-4931/$ see front matter 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.msec.2013.03.018perties such as intenseuorescence life time,

tive for bulk production.In the quest of exploring natural precursors for the synthesis ofuorescence, enhanced photostability, long1. Introduction

Optical labeling using conventionweak photostability and attenuatedthe other hand, quantum dots (QDs),properties have gained signicant fa[2]. The attributes which make QDsrochromes suffer fromcence intensity [1]. Ontheir appreciable opticaldiagnosis and labelingandidates for biological

shows red color [16]. There are very few reports on the synthesis ofC-dots using natural plant materials as precursors. Recently, C-dotwas synthesized using orange juice [17], Jaggery, Bread and Sugar[18]. Highly uorescent C-dots at 28 2 C were synthesized usingsugarcane juice followed by size dependent separation using sucrosedensity gradient centrifugation [19]. These C-dots being made fromnatural materials become exceptionally biocompatible and cost effec-TurbostraticBiocompatibilityMDCK cellsGreen synthesis of biocompatible carbonTrapa bispinosa peel

Ashmi Mewada, Sunil Pandey , Sachin Shinde, NeeraMaheshwar Sharon, Madhuri Sharon NSN Research Center for Nanotechnology and Bio-Nanotechnology, Ambernath, MS India

a b s t r a c ta r t i c l e i n f o

Article history:Received 17 January 2013Received in revised form 8 March 2013Accepted 11 March 2013Available online 26 March 2013

Keywords:C-dotsTrapa bispinosa

We are reporting highly econdots (C-dot) using Indianwa90 C. C-dots ranging from 5UV-light (ex = 365 nm). Uabsorption of C-dots betweenexhibited characteristic emisanalyzed using X-ray diffractof the C-dots was found to b

j ourna l homepage: wwwtion of candle soot [14].ned by different surfaceith octadecylamine ex-

hestrated with diamond

1 7738498299.dey),

rights reserved.ts using aqueous extract of

Mishra, Goldie Oza, Mukeshchand Thakur,

ical plant basedmethod for the production of luminescent water soluble carbonplant Trapa bispinosa peel extract without adding any external oxidizing agent at10 nm were found in the solution with a prominent green uorescence underis spectra recorded at different time intervals (30120 min) displayed signature0 and 600 nm. Fluorescence spectra of the dispersion after 120 min of synthesispeaks of C-dots when excited at 350, 400, 450 and 500 nm. C-dots were further(XRD), Raman Spectroscopy and Thermo-Gravimetric Analysis (TGA). Structureurbostratic when studied using XRD. C-dots synthesized by our method were

nd Engineering C

sev ie r .com/ locate /msec2. Materials and methods

All the experiments were performed using nano-pure water. Trapabispinosa was procured from the local market and soaked in coldwater for 30 min. 50 grams of the peel was crushed in 500 ml ofdistilled water and centrifuged to obtain clear light pink extract. Forsynthesis of C-dots, 100 ml of T. bispinosa peel extract was reuxedfor 2 h at 90 C till the solution becomes greenish brown. Resulting

intensities of the spectra with respect to excitation wavelengths.HRTEM image (Fig. 3a) shows the presence of spherical C-dots

ranging from 5 to 10 nm. Size distribution of the nanoparticles isdisplayed in the histogram (Fig. 3b).

Fig. 4a displays the XRD image of C-dots showing the presence ofintense peak at 2 = 24.7 and a weak peak at 2 = 43.3 that are

Fig. 2. Fluorescence spectroscopy of C-dots excited at different wavelengths. Inset

2915A. Mewada et al. / Materials Science and Engineering C 33 (2013) 29142917solution was centrifuged at 5000 rpm for 20 min and suspended with5 ml 1 N NaOH to solubilize C-dots and enhance the uorescenceability. In order to purify C-dots, 3 ml of the above solutionwas dialysed(MW = 12kD) against nanopure water for 24 h. Clear yellowish sus-pension of C-dot was observed which exhibited intense green uo-rescence under UV-light. Quantum yields (QY) of the C-dots werecalculated using following equation:

s RAR R n2s IsAs s n2RIR

1

where, S and R are quantum yields of sample and referencerespectively; AR (R) and As (s) are the absorbance of the referenceand sample at excitation wavelengths; nS and nR are refractive indi-ces of the sample medium and reference medium respectively;IS and IR are the integrated uorescence intensities of sample andreference respectively. Due to highly stable luminescent propertiesof Quinine sulfate ( = 0.54) was used as a reference to determineQYs [20] of C-dots excited at 350, 400, 450 and 500 nm.

2.1. Characterization

Spectral properties of the C-dots were studied by UVvis Spectros-copy (Lambda-25, Perkin Elmer, USA) and Fluorescence spectroscopy(Perkin Elmer, USA) in a standard quartz cuvette. 350, 400, 450 and500 nm were selected as excitation wavelengths.

Fourier Transform Infra-Red (FTIR) Spectroscopic studies wereperformed within the spectral window 500 to 4000 cm1. HighResolution Scanning Electron Microscopy (HRTEM) [Carl Ziess, GmbH,Germany] studies were performed by coating aqueous sample ofC-dots puried by dialysis, on carbon coated formwar.

Crystallinity of C-dots was studied using X-ray diffraction (XRD)[Phillips, The Netherlands]. For XRD analysis, samples were dried ona glass coverslip. Raman spectra were recorded using a Jobin-YvonLabram spectrometer. Samples were excited using lasers (632.8, 532and 488 nm) with a spectral resolution of b1.5 cm1. All the spectrawere initially baseline corrected with 3rd order polynomial and nor-malized to the max of the peak intensity.

For Thermo-Gravimetric Analysis (TGA) analysis, 20 mg of samplewas placed in the TGA cell [Perkin Elmer, Diamond, USA] and heatedto 830 C at a heating rate of 20 C min1 in an atmosphere of N2 gas.

2.2. Cytotoxicity studies

MTT assay was used to study the cytotoxic effects of C-dots onMDCK cells. This assay is based on the mitochondrial enzyme cata-lyzed conversion of pale yellow MTT to violet color formazan crystals.Cells were seeded (5 105/ml) in 96 well plates and incubated over-night at 37 C and 5% CO2. Medium was then replaced with C-dots(14 g/ml) and incubated further for 48 h. After incubation C-dot solu-tion was replaced with MTT solution (200 g/ml) and cells were incu-bated for 2.5 h at 28 2 C to initiate the formation of formazan. Thissolution was replaced with 150 l of Dimethyl Sulfoxide (DMSO)[Sigma, USA] and complex wasmixed gently to dissolve formazan crys-tals. Finally, the dissolved formazan in DMSO was transferred to fresh96well plates and read onmicroplate reader [Thermo, USA] at 570 nm.

3. Results and discussion

The color of T. bispinosa peel extract transformed to greenish brownafter heating at 90 C for 120 min due to the formation of C-dots. Thisis mainly due to the thermal oxidation of precursors present in the ex-tract of T. bispinosa. Brown color of the solution appeared extremelyuorescent underUV-light (ex = 365 nm) exhibiting deep greenuo-rescence (inset of Fig. 1). Fluorescence in C-dots may appear due to the

recombination of electronhole pair from impurity atoms and oxygenbearing functional groups [2]. However, the exact mechanism of originof multicolored luminescence is still a brain child.

To explore the optical characteristics of biologically synthesizedC-dots, UVvis spectroscopy (Fig. 1) of the samples recorded at theinterval of 30 min shows continuous red shift followed by blue shiftafter 120 min. Initially, a broad peak and a shoulder at 538 and875.61 nm were observed indicating the slow formation of C-dot asper previous studies. After 60 min, there was a slight narrowing andred shift (from 538 to 541 nm) indicating the controlled formation ofmonodispersed C-dots. Finally after 120 min, a peak and shoulder at536 and 907 nm respectively were observed. There was a blue shift of2 nm after 120 min explaining the stabilization of the nanoparticlesand/or decrease in the size of the C-dots.

Foruorescence studies (Fig. 2), C-dots collected after 120 minwereconsidered due to excellent uorescence under UV-light indicatingcomplete formation. Typical excitation based emission spectra of the so-lutions recorded at different excitation wavelengths 350, 400, 450 and500 nm displayed unique optical properties of C-dots as per previousstudies [2]. Intensities of the C-dotswere found to be increasedwith re-spect to increase in the excitation wavelength. Maximum intensity wasachieved at excitation wavelength of 450 nm. Inset of Fig. 2 shows the

Fig. 1. UVvis spectroscopy of C-dots representing characteristic absorption between450 and 650 nm. Inset shows intense green color under UV-light (ex = 365 nm) ofC-dots.shows intensity of the C-dots with respect to excitation wavelengths.

rticl

2916 A. Mewada et al. / Materials Science and Engineering C 33 (2013) 29142917Fig. 3. (a) HRTEM of C-dot displaying the presence of ultra-small paassigned to (002) and (101) diffraction patterns of graphitic carbonrespectively. Former peak corresponds to the interlayer spacing of~3.77 which is slightly more than the spacing between (002)planes in bulk graphite (3.44 ). These ndings explain turbostraticcarbon structures. Pan et al. explained graphitic nature of the C-dotswith an inner layer spacing of ~4.12 which is in strong agreementwith our ndings [21].

Typical Raman spectra in Fig. 4b shows the graphitic natureof C-dot. Sharp and intense Raman peak of G-band observed at1578 cm1 with respect to feeble peak of D-band at 1331 cm1

shows the presence of pristine carbon nanomaterials in the formof C-dots [22]. An additional peak at 2654 cm1 explains the SP2

hybridization pattern. It is the 2nd order two phonon process butsometimes clearly seen in Raman spectra. A ratio of intensities ofID/IG was calculated to be 0.59 which denotes the purity of theC-dots.

FTIR spectra (Fig. 5a) shows the functional groups associatedwith the surface of C-dots. A strong absorption at 2925 cm1 and

Fig. 4. (a) XRD of the C-dots showing typical Bragg

Fig. 5. (a) FTIR and (b) TGA analyses of C-dotses and (b) histogram depicting size distribution of the particle size.2860 cm1 is assigned to \C\H stretching which may arise due tomethyl or methylene groups associated with the aliphatic hydrocar-bons present in the extract of T. bispinosa peel. A weak absorption at3394 cm1 is due to O\H stretching. An overtone at 1648 cm1 isdue to \C_C\ stretching. The presence of such functional groupsexplains the functionalization of C-dots containing \CH3 and \OHwhich can be used as linkers for the attachment of the therapeuticmoieties such as drugs for targeted delivery to diseased cells.

TGA measures the amount and rate of change of the weight ofmaterial (weight loss) with respect to temperature under controlledenvironment. Weight loss at different temperatures indicates the

's reections and (b) Raman spectra of C-dots.

synthesized using T. bispinosa peel extract.

Table 1QYs of the C-dots synthesized using T. bispinosa at different excitation wavelengths.

EW (nm) 350 400 450 500QYs 0.1 0.24 1.2 0.87

EW excitation wavelength.

ce

2917A. Mewada et al. / Materials Science and Engineering C 33 (2013) 29142917composition as well as thermal stability of the complex. TGA analysissuggests that C-dots were stable at 110 C and loss of weight resultedafterwards (Fig. 5b). This may be due to the degradation of associatedchemical moieties with the C-dots. There is another loss of weightat 444 C which may be due to those species which are anchoredstrongly with C-dots via covalent linkages. This conrms surfacefunctionalization of C-dots which confers specic uorescence [14].QYs of the C-dots calculated at all the excitation wavelengths aresummarized in Table 1. It was found to bemaximum for C-dots excitedat 450 nm. QYs of 1.2 at 450 nm can be enhanced by further surfacefunctionalization with passivation agents.

C-dots were found extremely biocompatible againstMDCK cells. Atall the concentrations (14 g/ml) the survival of the cells was foundto be more than 80% (Fig. 6a). At 1 g/ml, no killing in the cells wasobserved whereas, at 4 g/ml 80.32% cells were found to be alive.This indicates the exceptional biocompatibility of the C-dots fornovel biological applications. There can be plethora of ways, C-dotscan confront with MDCK cells. Some of the possible mechanisms mayinvolve receptor mediated endocytosis, direct anchoring to cell mem-branes. Killing of the cells to some extent can be due to obstructionof the channel proteins or blocking transporters which mediate entryof vital metabolites. However, any concrete mechanism involving suchinteractions is not yet proposed.

Fig. 6b displays the Fluorescence microscopic image of C-dots.The fundamental property of intense green uorescence exhibitedby C-dots in solution can be exploited for cellular imaging.

4. Conclusion

T. bispinosa peel extract was found to be an excellent source formass production of luminescent C-dots without adding any externalagents such as ethanol. C-dots formed using this method exhibitsideal uorescent properties and quantum yield. C-dots, being purely

Fig. 6. (a) Cytotoxicity of c-dots using MDCKbiological in origin can be used for biological applications such asdelivery of active pharmaceutical ingredients and genes inside thecells.Acknowledgment

Authors wish to acknowledge the nancial support provided bythe authorities of SICES, Ambernath and specially Mr. K.M.S. Nair andMr. K.M.K Nair. We give special thanks to Dr. Lala of UGC-DAE, Indorefor doing TEM analysis.

References

[1] H. Mattoussi, J.M. Mauro, E.R. Golaman, G.P. Anderson, V.C. Sundar, F.V. Mikulec,M.G. Bawendi, J. Am. Chem. Soc. 122 (2000) 1214212150.

[2] S.N. Baker, G.A. Baker, Angew. Chem. Int. 49 (2010) 67266744.[3] H.B. Li, Y. Zhang, X.Q. Wang, Sens. Actuators B 127 (2007) 593597.[4] W.B. Tan, N. Huang, Y. Zhang, J. Colloid Interface Sci. 310 (2007) 464470.[5] S.J. Cho, D. Maysinger, M. Jain, B. Roder, S. Hackbarth, F.M. Winnik, Langmuir

23 (2007) 19741980.[6] Y.P. Sun, K.F. Fu, Y. Lin, W.J. Huang, Acc. Chem. Res. 35 (2002) 10961104.[7] M. Hasheminezhad,H. Fleischner, B.D.McKay, Chem. Phys. Lett. 464 (2008) 118121.[8] J.C.G. Esteves da Silva, H.M.R. Goncalves, Trends Anal. Chem. 30 (2011) 13271336.[9] Y.P. Sun, B. Zhou, Y. Lin, W. Wang, K.A.S. Fernando, P. Pathak, M. Meziani, B.A.

Harruff, X. Wang, H. Wang, P.G. Luo, H. Yang, M.E. Kose, B. Chen, L.M. Veca, S.Y.Xie, J. Am. Chem. Soc. 128 (2006) 77567757.

[10] L. Cao, X. Wang, M.J. Meziani, F. Lu, H. Wang, P.G. Luo, Y. Lin, B.A. Harruff, L.M.Veca, D. Murray, S.Y. Xie, Y.P. Sun, J. Am. Chem. Soc. 129 (2007) 1131811319.

[11] B.R. Selvi, D. Jagadeesan, B.S. Suma, G. Nagashankar, M. Arif, K. Balasubramanyam,M. Eswaramoorthy, T.K. Kundu, Nano Lett. 8 (2008) 31823188.

[12] A.B. Bourlinos, A. Stassinopoulos, D. Anglos, R. Zboril, V. Georgakilas, E.P. Giannelis,Chem. Mater. 20 (2008) 45394541.

[13] Q.L. Zhao, Z.L. Zhang, B.H. Huang, J. Peng, M. Zhang, D.W. Pang, Chem. Commun.(2008) 51165118.

[14] H. Liu, T. Ye, C. Mao, Angew. Chem. Int. 46 (2007) 64736475.[15] V.N. Mochalin, Y.J. Gogotsi, J. Am. Chem. Soc. 131 (2009) 45944595.[16] A. Gruber, A. Drbenstedt, C. Tietz, L. Fleury, J. Wrachtrup, C. von Borczyskowski,

Science 276 (1997) 20122014.[17] S. Sahu, B. Behera, T. Maiti, S. Mohapatra, Chem. Commun. 48 (2012) 88358837.[18] sk Palashuddin, A. Jaiswal, A. Paul, S. Ghosh, A. Chattopadhyay, Sci. Rep. 2 (2012)

383388.[19] S. Pandey, A.Mewada, G. Oza, M. Thakur, N.Mishra, M. Sharon, M. Sharon, Nanosci.

Nanotechnol. Lett. 5 (2013) 17.[20] W.H. Melhuish, J. Phys. Chem. 65 (1961) 229235.[21] D.Y. Pan, J.C. Zhang, W.Q. Shen, Z.W. Zhang, Y.G. Fang, M.H. Wu, New J. Chem.

34 (2010) 591593.

lls and (b) uorescence microscopic image.[22] M.S. Dresselhaus, A. Jorio, M. Hofmann, G. Dresselhaus, R. Saito, Nano Lett. 10 (2010)751758.

Green synthesis of biocompatible carbon dots using aqueous extract of Trapa bispinosa peel1. Introduction2. Materials and methods2.1. Characterization2.2. Cytotoxicity studies

3. Results and discussion4. ConclusionAcknowledgmentReferences