Electronic Supporting Information

A Green route towards Highly Photoluminescent and

Cytocompatible Carbon dot synthesis and its separation using
sucrose density gradient centrifugation
Goldie Oza*, Kusum Oza, Sunil Pandey, Sachin Shinde, Ashmi Mewada, Mukeshchand

Thakur, Maheshwar Sharon and Madhuri Sharon

Department of Electrical Engineering (SEES) CINVESTAV-IPN, Avenida IPN 2508, San

a

Pedro Zacatenco, Mexico D.F, b Nagarjuna University, Andhra Pradesh , India, cNational Sun

Yat Sen University, 80424 lotus sea, Gushan District, Kaohsiung City, Taiwan. dNagoya

Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi, 466-8555 Japan, e N.

Shankaran Nair Research center for Nanotechnology and Bionanotechnology, Jambhulphata,

Ambernath, MS, India.

1. Material and Methods

1.1 Materials: Reagent-grade Sodium Hydroxide and Sodium borohydride were purchased

from Aldrich and used as received. High-purity water with the resistivity of greater than 18

M·cm-1 was used in the experiments.

1.2 Methods

1.2.1 Synthesis of C-dots: 10ml of Citrus limone juice was extracted and then filtered using

muslin cloth. This extract was then centrifuged at 5000 rpm for 15 mins NaOH solution (5M)

in the ratio of 1:3 (i.e. 1 part of extract and 3 parts of NaOH). The whole solution was kept in

an ultrasonicator (20 kHz) for 1 hour. When the solution turned from pale turbid yellow

colour to brown colour, the reaction was stopped and checked for fluorescence under UV

tube of 365 nm. Strong reducing agent sodium borohydride (2ml of 10mM) was then added

to this mixture to reduce C-dots, thus enhancing the fluorescence property of the solution.
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1.2.2 Density gradient centrifugation of C-dots: In sucrose density gradient centrifugation

method, sucrose gradient is created in a 50-mL centrifuge tube by sequentially layering 1ml

each of 100% -50% (w/v) sucrose solutions from bottom to top. Onto the top of such a

sucrose gradient solution, water- soluble C-dot mixture was finally dropped. This sample tube

was then loaded in a fixed angle rotor and centrifuged at 5000 rpm for 1 hr.

1.2.3 Confocal Microscopy of Hep-2 cells on exposure to Carbon-dots from both the

fractions F1 and F2

Hep-2 cells were cultured in 24 well plates at 370C in 5% CO2 incubator. The culture

medium was Dulbecco`s Modified Eagle Medium (containing 10% fetal bovine serum and

1% penicillin as well as streptomycin). The culture medium was changed after every three

days. For sub-culturing, the cells were washed twice with PBS and detached by treating with

Trypsin EDTA at 370C for 10 minutes. The cells were harvested by centrifugation (1,600

rpm, 5 min.) and resuspended in the culture medium. Approximately 6×10 6 cells were

cultured for 48 hours in a 24 well plate with a polylysine-treated cover slip with 1mL media

and then were mixed with 10mM concentration of of both F1 and F2 fractions of carbon-dot

solution and were incubated for 10 hours. After incubation for 10 hours, the medium was

aspirated and then 0.5ml of 2% formaldehyde was added for 15 minutes. This was then

followed by washing with phosphate buffered saline. Controls without any labelling were

also used. The cells were imaged with a Carl Zeiss Apotome Imager Z1 microscope. The F1

fraction was excited by 488nm wavelength and was detected with 530nm long pass filter,

while F2 was excited with 458nm wavelength and detected with 475nm long pass filter.

1.2.4 Cytotoxicity assay

The Hep-2 cells were treated with different concentration of carbon dots ranging from 2-

100mM for both F1 and F2 in 24-well plate and were incubated for 24 hours. The cells

attached to each plate were then treated with freshly prepared methylthiazolyldiphenyl-
2
tetrazolium bromide (MTT) solution (5 mg/mL). The cells were then again incubated for 3

hrs for the formation of violet-coloured formazan. The formazan was then dispersed in a 50%

DMSO solution and the absorbance was then measured with an ELISA Microplate reader.

The cell viability was measured considering 100% viability for the control sample without

any carbon dots in it.

2. Characterization

2.1 High Resolution-Transmission electron microscopy (TEM): The particle diameter and

lattice fringes were examined with a JEOL JEM-2100 transmission electron microscope. A

drop of carbon dot aqueous solution was placed on a copper grid and then dried before

transferring into the TEM sample chamber.

2.2 Spectroscopy: Fourier-transform infrared (FT-IR) spectra were recorded on a Nicolet

6700 FT-IR spectrometer. Raman studies were performed using a Horiba HR 800 Raman

system equipped with a 514.5 nm laser. UV-Vis absorption spectra were recorded by a UV-

Vis spectrometer (Lambda 25, Perkin-Elmer). Fluorescence measurements were done by

using Hitachi 7000 Fluorescence spectrophotometer. XRD patterns were determined from

Regaku D/max/2500 instrument using Cu-Kα radiation. Carbon content was obtained using

2400 series II Elemental analysis. Zeta potential was derived from Zetasizer Nano ZS90

(Malvern instruments) at a wavelength of 633 nm with a 4.0 mW, solid-state He–Ne laser at a

scattering angle of 90° at 25°C.

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3. Results

Table S1. A comparative Quantum yield report from different methods

Cdot synthesis Method Quantum yield (%) References

Electrochemical method 0.06 Zhou et al,

2007

Heating under reflux in nitric acid 1.15 Wang et al,

2011

Microwave-assisted heating under reflux 1.91 Wang et al,

2011

Microwave-hydrothermal method 2.72 Wang et al,

2011

Solvothermal method 7% Wang et al

2013

Ultrasonic method, fraction F2 15% In the present

work

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Fig.S2 X-ray diffraction pattern of Carbon dot showing lattice planes at (002), which is
similar for both the fractions

Fig. S3 Raman spectrum showing both D and G band, which is again similar in both the
fractions