Materials Science and Engineering C 38 (2014) 20–27

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

One-pot green synthesis of carbon dots by using Saccharum officinarum

juice for fluorescent imaging of bacteria (Escherichia coli) and yeast
(Saccharomyces cerevisiae) cells
Vaibhavkumar N. Mehta a, Sanjay Jha b, Suresh Kumar Kailasa a,⁎
a
Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007, India
b
Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, 395007, India

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

Article history: We are reporting highly economical plant-based hydrothermal method for one-pot green synthesis of water-
Received 12 November 2013 dispersible fluorescent carbon dots (CDs) by using Saccharum officinarum juice as precursor. The synthesized CDs
Received in revised form 14 December 2013 were characterized by UV-visible, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering
Accepted 21 January 2014
(DLS), high-resolution transmission electron microscopic (HR-TEM), and laser scanning confocal microscopic
Available online 29 January 2014
techniques. The CDs are well dispersed in water with an average size of ~3 nm and showed bright blue fluores-
Keywords:
cence under UV-light (λex = 365 nm). These CDs acted as excellent fluorescent probes in cellular imaging of bac-
Saccharum officinarum juice teria (Escherichia coli) and yeast (Saccharomyces cerevisiae).
Fluorescence microscopy © 2014 Elsevier B.V. All rights reserved.
DLS
TEM
Bacteria and yeast cells

1. Introduction emission. Literature survey reveals that a variety of synthetic routes

The monitoring of the biomolecules of real-time dynamics plays a
key role in the translocation of molecules and interacts with partners
in cell biology [1]. Recent breakthroughs of nanoscience in cell biology
have provided new approaches to address and to solve problems in bi-
ological assays and in clinical diagnostics. Furthermore, the evaluation
of subcellular structures by light microscopy is important in many fields,
including cell biology and pathology diagnostics [2,3]. In this connec-
tion, two-photon fluorescence nanomaterials have been integrated in
light microscopic methods for the visualization of cells in tissues with
high resolutions [4,5]. Among the fluorescent nanomaterials, fluores-
cent carbon dots have received considerable interest in the field of opto-
electronic devices and biocompatible imaging probes [6–8]. CDs are the
new class of small oxygenous carbon nanoparticles with sizes b 10 nm,
which exhibit excitation wavelength-dependent photoluminescence
properties based on their displaying size. Importantly, CDs have several
advantages over fluorescent semiconductor nanomaterials, such as ex-
cellent optical properties, high chemical stability, and low environmen-
tal hazard, making them promising light-emitting materials to visualize
the cell in vitro and in vivo. The CDs showed superiority in chemical in-
ertness and lower toxicity than the traditional quantum dots (QDs) [9].
However, the preparations of CDs involve the use of chemicals and sev-
eral experimental setups to synthesize the CDs with multi-color
⁎ Corresponding author. Tel.: +91 261 2201730; fax: +91 261 2227334.
E-mail addresses: sureshkumarchem@gmail.com, skk@ashd.svnit.ac.in (S.K. Kailasa).
such as arc-discharge [10], electrochemical oxidation [11,12], hydro-
thermal [13], laser ablation [14], microwave heating [15,16], combus-
tion [9,17], and supported-synthesis [18] have been developed for the
preparation of fluorescent CDs and used as imaging probes for metal
ions and biomolecules. However, these approaches require expensive
precursors and involve specific/sophisticated experimental setup/
condition and complex or post-treatment processes for the synthesis
of fluorescent CDs. As cost is an important criterion in the success of
fluorescent CDs, new green approaches are essentially needed to pro-
duce fluorescent CDs without the use of organic chemicals and tedious
experimental setups.
In recent years, several researchers have devoted their time on the
preparation of fluorescent CD emitters without the use of organic
chemicals in a single step, which is called as green chemistry concept
[19,20]. To support this assumption, several green synthetic approaches
have been developed for the preparation of CDs by using inexpensive
renewable resources as the precursor. Briefly, Han's and Zou's groups
developed green synthetic approach for the synthesis of water-soluble
fluorescent CDs by using watermelon peel as raw resource at low-
temperature carbonization and used as live cell imaging probes [21].
Lu and co-workers [22] reported a simple, economical, and green pre-
parative hydrothermal strategy for synthesis of water-soluble, fluores-
cent carbon CDs with a quantum yield of ~ 6.9% by using pomelo peel
as a carbon source and used as fluorescent probes for detection of
Hg2+. The same group described the use of flour as the carbon source
for the microwave-assisted rapid green synthesis of photoluminescent

0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.msec.2014.01.038
 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27
CDs (1–4 nm) and used as a fluorescent sensor for the detection of metal
ions [15]. Pandey's and Sharon's teams described the potential use of
Indian water plant Trapa bispinosa peel for the preparation of lumines-
cent water-soluble CDs (5–10 nm) with exceptionally biocompatible
against MDCK cells [23]. The photoluminescent CDs with (26% quantum
yield) have been prepared in a single step by the hydrothermal treat-
ment of orange juice and used as fluorescent probes for cellular imaging
[24]. Recently, luminescent CDs were prepared by using renewable re-
sources such as jaggery, bread, and sugar at room temperature [25].
Apart from these methods, Li's group described a simple approach for
the synthesis of fluorescent CDs by using gelatin as carbon source in the
hydrothermal treatment and used as biocompatible materials for the vi-
sualization of cells [26]. Wang et al. [27] developed a one-pot fabrication
approach for the synthesis of fluorescent CDs by using ascorbic acid at
90 °C. Koshizaki and co-workers [28] prepared photoluminescent CDs
by using laser rapid passivation technique with ordinary organic solvents
as carbon source. Similarly, Chang's group described a simple one-pot
hydrothermal method for the preparation of photoluminescent CDs by
using low-cost organic compounds as carbon sources and used as fluo-
rescent probes for imaging of MCF-10A and MCF-7 cells [29]. Recently,
Wei et al. [30–32] described a series of facile chemical routes for
the preparation of highly water-dispersible fluorescent organic nano-
particles and their uses as promising fluorescent bioprobes for the visu-
alization of cells in biological samples. With the inspiration of these
achievements, we report a novel green chemistry synthetic approach
for the preparation of water-soluble fluorescent CDs (~3 nm) in a single
step by using the Saccharum officinarum (sugar cane) juice as a rich car-
bon source. The synthesized CDs were characterized by UV-visible, fluo-
rescence, FT-IR, 1H- and 13C-NMR, DLS, and HR-TEM techniques. The
synthesized CDs are well dispersed in water and exhibited strong visible
fluorescence and up-conversion photoluminescence. These CDs acted
multicolor-emitting probes for cell imaging of bacteria (Escherichia coli)
and yeast (Saccharomyces cerevisiae).
2. Materials and methods
2.1. Chemicals and materials
S. officinarum juice was purchased (rupees 20 per 500 mL) from the
local market of Surat, India. Ethanol, dichloromethane, acetone, sodium
chloride, and glucose were purchased from Merck Ltd., India. All
chemicals were of analytical grade and used without further purifica-
tion. Milli-Q-purified water was used for sample preparations.
2.2. Synthesis of carbon dots from Saccharum officinarum juice
The hydrothermal method was used for the preparation of water-
dispersible CDs by using S. officinarum juice as carbon source. The
water-dispersible fluorescent CDs were synthesized according to the re-
ported method with minor modification [24]. Briefly, 350 mL of
S. officinarum juice was mixed with 150 mL ethanol, and then the mix-
ture was transferred into a 700-mL Teflon-lined stainless-steel auto-
clave. The reaction mixture was heated at constant temperature
(120 °C) for 180 min until a dark brown solution formed. The autoclave
was allowed to cool down naturally. The resulted dark brown solution
was washed with dichloromethane to remove unreacted organic moie-
ties. The aqueous solution was collected and centrifuged at 5000 rpm for
20 min to separate the less-fluorescent deposit. Excess acetone was
added to the upper brown solution and centrifuged at 13,000 rpm for
15 min to obtain highly fluorescent CDs with ~2.5–3.0 nm. In order to
know the optimum temperature for high efficient fluorescent CDs, we
prepared the water-dispersible CDs at different temperatures 100 °C,
120 °C, and 140 °C, and the obtained CDs solutions were confirmed by
UV-visible absorption and fluorescence emission spectra.
21
2.3. Cell labeling assay
E. coli DH5α cells were grown in Luria–Bertani (LB) medium
(10 mg/mL Tryptone, 5 mg/mL Yeast Extract and 10 mg/mL NaCl
pH 7.0) at 37 °C, 180 rpm. The diploid strain S. cerevisiae were grown
in yeast extract-peptone-dextrose (YPD) medium (10 mg/mL yeast ex-
tract, 20 mg/mL Bacto Peptone, 20 mg/mL glucose, pH 6.5 ± 0.2) at
30 °C. The cultures were shaken at 150 rpm, and growth was monitored
by measuring the absorbance at 600 nm. E. coli and S. cerevisiae cells
were collected from the middle of the exponential growth phase and
used for confocal microscopy analysis. For CD internalization studies,
cells were fixed and the fixation was performed with 70% (v/v) ethanol
at 4 °C for 5 min. The fixed cells were resuspended in 100 mM of phos-
phate buffer containing CDs (CDs: 40 μg/mL) at room temperature for
10 min. The stained cells were washed twice and subjected to confocal
microscopy. Cell imaging was done under a Carl Zeiss 510 LSM laser
scanning confocal microscope with a laser excitation of 405, 488, and
561 nm, and fluorescence was collected in blue, green, and red region.
2.4. Cytotoxicity study of CDs
The growth curve of E. coli and S. cerevisiae was plotted by growing
the cells in modified M9 and YNB (Yeast Nitrogen Base) medium in
150 mL Erlenmeyer flasks at 37 °C and 28 °C for 12–16 h. To these flasks,
0, 10, 20, 40, 100, 200, and 400 mg/mL of CD concentrations were added
and incubated by using orbital shaker at 180 rpm. In order to know cell
viability, a 2-mL culture broth was collected at different time intervals
(0–24 h) and measured for their optical density (at 600 nm) using a
spectrophotometer (CARY 50 UV, Australia).
2.5. Quantum yield measurement
The quantum yield of the CDs was calculated by measuring the fluo-
rescence intensity in aqueous dispersion by using the following equa-
tion [33],
I CD AR η2CD
Q CD ¼ Q R    ð1Þ
IR ACD η2R
where Q is the quantum yield, I is the intensity of luminescent spectra, A
is the absorbance at exited wavelength, and η is the refractive index of
the solvent used, using quinine sulfate (quantum yield 54%) in 0.1 M
H2SO4 solution as the reference. The subscripts CD for carbon dots and
R for reference are used in this equation. The quantum yield of CDs
was measured at an excitation wavelength of 360 nm, and yield was
found to be 5.76%.
2.6. Instrumentation
UV-visible spectra were measured by using Maya Pro 2000
spectrophotometer (Ocean Optics, USA) at room temperature. The fluo-
rescence spectra were recorded using an RF-5301 PC Shimadzu
spectroflourometer. Fourier transform infrared (FT-IR) spectra were re-
corded on a Perkin Elmer (FT-IR spectrum BX, Germany). The morphol-
ogy and the microstructure of the CDs were examined by HR-TEM on
a JEOL 3010 with an accelerating voltage of 200 kV. The samples
for HR-TEM were made by dropping an aqueous solution onto a
300-mesh copper grid coated with a lacy carbon film. The x-ray diffrac-
tion (XRD) profiles of the prepared samples were recorded on a Rigaku
diffractometer (Rigaku, Japan) equipped with graphite monochromatized
CuKα (λ = 0.15405 nm) radiation in the range from 10° to 70°. DLS mea-
surements were performed by using Zetasizer Nano ZS90 (Malvern, UK).
22 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27

Hydrothermal 13000 rpm

o
120 C for 180 min Extraction Centrifuge
with DCM
Crude CDs
CDs in CDs in water CDs under UV
aqueous layer light at 365 nm

Fig. 1. Schematic representation for the formation of carbon dots through the hydrothermal treatment of Saccharum officinarum juice.

3. Results and discussion
3.1. Characterization
The multicolor emission CDs were prepared by using different car-
bon source materials, nucleation, and growth conditions. The carbon
source material and conditions play a key role for the preparation of
CDs with tunable emission and with high quantum yield. The color of
S. officinarum juice was changed from green to dark brown after heating
the juice at different temperatures 100 °C, 120 °C, and 140 °C for
180 min, confirming the formation of CDs. Fig. 1 shows the schematic
representation for the preparation of CDs by using S. officinarum
juice as carbon source. The carbonization of S. officinarum juice was
performed at different reaction temperatures from 100 °C to 140 °C.
To confirm the best optimum temperature, the obtained CDs were
placed on UV light and irradiated at excitation wavelengths 254, 302,
and 365 nm (Fig. 2). It can be observed that the CDs showed intense
bright blue color emission only at UV light excitation wavelength
365 nm, and the CDs did not exhibit any emission at excitation wave-
lengths 254 and 302 nm. As shown in Fig. 2, at 120 °C, carbonization
solution was well dispersed with good brown color and exhibited
bright blue color emission under UV light at the excitation wavelength
of 365 nm compared with carbonizations at 100 °C and 140 °C,
confirming that 120 °C is the best temperature for the preparation of
water-dispersible CDs with bright blue emission. Fig. 3 shows the UV-
visible absorption and emission spectra of CDs (1.0 mg/mL). The CDs

100 120 140 100 120 140

Temperature (oC) Temperature (oC) at λex 254 nm

100 120 140 100 120 140

Temperature (oC) at λex 302 nm Temperature (oC) at λex 365 nm

Fig. 2. Photographic images of synthesized CDs at different temperatures (100 °C, 120 °C, and 140 °C) and their fluorescence properties under UV light excitation wavelengths 254, 302,
and 365 nm.
 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27 23

474 nm
UV-visible
FL emission
We also measured the UV-visible and fluorescent spectra of CDs at
2.0 20 different carbonization temperatures (100 °C, 120 °C, and 140 °C). The
well-water-dispersed CDs exhibit bright blue emission under UV light
370 nm
(λex = 365 nm), which could be easily observed with the naked eye

Fluorescence intensity
1.5 15 (Inset in Fig. 3). The water-dispersed CDs showed a strong fluorescence
Absorbance

emission band at 474 nm by exciting the CDs solution at 390 nm, which
confirms that the formation of fluorescent CDs by using S. officinarum
1.0 10 juice as carbon source in hydrothermal method. Supporting Information
of Fig. S1 shows the UV-visible spectra of CDs at different temperatures
of 100 °C, 120 °C, and 140 °C, respectively. Supporting Information of
0.5 5 Fig. S2 shows the fluorescence emission spectra of CDs at different tem-
peratures (100 °C, 120 °C, and 140 °C) under excitation wavelength
390 nm. It can be observed that the fluorescence emission intensity
0.0 0 was gradually increased with increasing temperature from 100 °C to
300 400 500 600 700 120 °C, after that temperature the fluorescence emission intensity of
Wavelength (nm) CDs was decreased. Supporting Information of Fig. S3 reveals that exci-
tation peaks are located at 390 and 410 nm, which indicates that the
Fig. 3. UV-visible absorption and fluorescence emission spectra (λex = 390 nm) of CDs emission may be related to two kinds of transitions [26]. The fluores-
(1.0 mg/mL) synthesized at 120 °C. Inset picture shows the carbon dots under daylight
cence behavior of CDs is attributed to the presence of surface energy
(left) and UV light at 365 nm wavelength (right).
traps, which results to generate different emission colors through the
surface passivation in nanosize CDs [14]. Therefore, we anticipate that
this characteristic feature plays a key role to use them as multicolor-
show the UV absorption peak at 370 nm, which is the characteristic emitting probes for cell imaging by using fluorescence microscope. Fur-
peak for CDs by carbonization of renewable carbon source. This absorp- thermore, the quantum yield of CDs is 5.67%, which is well comparable
tion peak confirms the carbon π system or the n–p* transition of the car- with the reported methods green synthetic approaches in the literature
bonyl [24,26,27]. [29,34,35].

a
COO-
-C-H

C-O
C=O C=C

-OH C-O-C

b
(002)
Intensity (a.u.)

(101)

2 Theta

Fig. 4. (a) FT-IR spectrum and (b) XRD pattern of CDs.

24 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27

We also studied FT-IR spectroscopy for the identification of function-
al groups present in CDs. As shown in Fig. 4a, the CDs exhibit peaks at
3379 and 2978 cm- 1 correspond to –OH and C-H groups stretching
and bending vibrations, which is attributed to the presence of carbohy-
drates (sucrose and glucose) in S. officinarum juice. The peaks at 1045
and 1644 cm−1 belong to the stretching vibration, indicating that the
existence of −C–O–C− and –C = C− groups. The characteristic absorp-
tion peak at 1235 cm− 1 was attributed due to the presence of –C–O
stretching. The absorption peaks at 1693 and 1376 cm−1 correspond
to –C = O vibration and symmetric carboxylate stretching. These are
confirming that the aldoses/ketoses are oxidized during the carboniza-
tion of S. officinarum juice. Furthermore, we also studied XRD for the
confirmation of structural properties of CDs. As shown in Fig. 4b, the
CDs have exhibited the intense peak at 2θ = 22.72º (002) and a weak
peak at 2θ = 42.60º (101), confirming the diffraction patterns of gra-
phitic carbon in sugar cane juice, which is strongly agreed with reported
methods [25–27]. Supporting Information of Figs. S4 and S5 show
the 1H- and 13C-NMR spectra of CDs. The peaks at 2.0–3.0 ppm
corresponded to methylene protons, and the peak at 8.2 ppm represents
the protons of OH in CDs solution. Similarly, the 13C-NMR spectrum of
CDs shows the peaks at 15–40 ppm correspond to aliphatic sp3 carbon
atoms in CDs. The peaks at 97–105 ppm are attributed to O − C* =
CH− and O − C = C*H− groups in CDs.
In order to investigate the morphology and average size of synthe-
sized CDs, we studied HR-TEM and DLS for the characterization of CDs.
Fig. 5a–b shows the typical HR-TEM images of CDs at 20 and 10 nm
scale bar, in which most of CDs are well dispersed in aqueous solution
with uniform in size and possessed a nearly spherical shape with an av-
erage size of ~3 nm. Fig. 5c shows the DLS data of CDs, in which CDs are
well monodispersed with an average hydrodynamic diameter of
~2.71 nm. These data are well agreed with the above HR-TEM images.
3.2. CDs as fluorescent imaging probes for bacteria (E. coli) and yeast
(S. cerevisiae)
Nanobiotechnologists have found ways to visualize cells in microor-
ganisms by using CDs as fluorescent imaging probes. The CDs are fluo-
rescent nanometer-scale crystals that consist of an electron-dense core
composed of carbon in order to facilitate bioconjugation. The CDs are be-
coming increasingly popular as cellular probes for light microscopic im-
aging because of their unique optical and physical properties. These
include high fluorescent quantum yield, resistance to photobleaching,
a large absorption cross section, and the ability to precisely tune the
fluorescence emission. To extend the application potential of CDs as
fluorescent imaging probes, we have investigated the CDs as in vivo
cell imaging probes for bacteria (E. coli) and yeast (S. cerevisiae).
Fig. 6 shows the laser confocal fluorescence microscopic images of
E. coli DH5α under different excitation wavelengths at 405, 488, and
561 nm laser in bright field with fluorescence mode and in only fluores-
cence mode by using CDs as fluorescent imaging probes. As shown in
Fig. 6b–d, the confocal images of bacteria cells show obvious blue,
green, and red emissions by using CDs (40 μg/mL) as fluorescent probe
under bright field with fluorescence mode. We collected optical images
at 10 μm in the z-direction at 408 nm (blue), 488 nm (green), and 561
nm (red) emission and images were shown in Fig. 6e–g. As depicted in
Fig. 6, strong blue, green, and red fluorescence of E. coli cells can be seen
after incubation with CDs for 1–6 h. Furthermore, we have also taken
the confocal images of E. coli DH5α under different excitation wave-
lengths at 405, 488, and 561 nm laser in the fluorescence mode (without
bright field) by using CDs as fluorescent imaging probes (Fig. 6e–g). These
images indicate that CDs are widely dispersed and appeared in the mem-
brane and cytoplasmic area of E. coli cells. Importantly, there are no CDs
were found in cell membrane at both imaging modes (Fig. 6b–g).

a b

20 nm 10 nm

c
Average hydrodynamic diameter: ~ 2.71 nm
Number (%)

Size (d.nm)

Fig. 5. HR-TEM images of carbon dots at different magnification (a) 20 nm and (b) 10 nm. (c) DLS measurement of CDs in aqueous solution.
 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27 25

a b

10 μm 10 μm

c d

10 μm 10 μm

e f

10 μm 10 μm

10 μm

Fig. 6. Confocal laser microscopic images of Escherichia coli cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at excitation
wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with E. coli at excitation wavelengths (e) 405 (blue), (f) 488 (green), and (g)
561 (red) nm, without bright field. Scale bar indicates 10 μm.

Fig. 7 shows the laser confocal fluorescence microscopic images of
yeast (S. cerevisiae) by using CDs (40 μg/mL) as fluorescent imaging
probes at 405, 488, and 561 nm. To this, we incubated yeast
(S. cerevisiae) cells in CDs for 1–6 h and monitored by confocal fluores-
cence microscopy. As shown in Fig. 7b–d, the strong blue, green, and red
fluorescence exhibited from CDs-labeled yeast cells upon the excitation
of CDs-conjugated cells at 405, 488, and 561 nm in bright-field
fluorescence emission mode, whereas no fluorescence was observed
from the control sample without the treatment of CDs. These results il-
lustrate that the CDs were effectively up taken to the cells, which results
a possible endocytosis mechanism through their homogeneous distri-
butions inside the cell [29]. As can be seen in Fig. 7b–g, more than 90%
of yeast cells are remained alive and exhibited intense blue, green, and
red fluorescence, which demonstrates that the prepared CDs are
26 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27

a b c d

10 μm 10 μm 10 μm 10 μm

e f g

10 μm 10 μm 10 μm

Fig. 7. Confocal laser microscopic images of Saccharomyces cerevisiae cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at
excitation wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with S. cerevisiae at excitation wavelengths (e) 405 (blue), (f) 488
(green), and (g) 561 (red) nm, without bright field. Scale bar indicates 10 μm.

exhibited beautiful fluorescence colors with low cytotoxicity. To further
confirm the localization of CDs inside the yeast nucleus, we have mea-
sured the confocal fluorescence microscopic images of yeast cells by
using CDs as imaging probes at fluorescence mode only (without bright
field) (Fig. 7e–g). It can be seen that CDs were preferentially well dis-
persed and located at the nucleus of yeast. These results successfully il-
lustrate that CDs possess good biocompatibility and can be effectively
applied multiple fluorescence emission for in vivo cell imaging and
a yeast cells. Fig. 8 shows the cytotoxicity study of CDs in E. coli and
0 mg/mL
10 mg/mL
OD
biological labeling. Importantly, it has been observed that these results
are quite different from the report of CdSe QDs as fluorescent imaging
probes for yeasts [36]. As comparison of CDs with the biosynthesized
CdSe QDs as fluorescent probes, the CDs acted as an effective fluorescent
probes for in vivo cell imaging of yeast as well as bacteria with intense
multiple colors (blue, green, and red). Based on the above observations,
we assume that the highly water-dispersible CDs (~3 nm) in the yeast
cells possibly involves extracellular growth, and a subsequent endocy-
tosis pathway is responsible for in vivo imaging.
Furthermore, we also studied the cytotoxicity of CDs in bacteria and
S. cerevisiae by using CD concentration ranging from 0 to 400 mg/mL.
These results indicate that CDs are exhibited non-toxicity up to
400 mg/mL for bacteria and yeast, which confirms that the CDs acted
as eco-friendly biological fluorescent-labeling probes for cellular and

20 mg/mL
40 mg/mL
in vivo imaging applications. It was found that 40 μg/mL of CDs is suffi-
100 mg/mL cient for the tagging of multiple cell of E. coli and S. cerevisiae. These re-
200 mg/mL sults indicate that the multicolor CDs derived from S. officinarum juice
400 mg/mL have exhibited very good biocompatibility and acted as nontoxic good
candidates for fluorescence imaging of wide variety cells (bacteria and
yeast), which are alternative to semiconductor nanocrystals.
Time (h)
4. Conclusions
b
In this paper, we described a novel, efficient and simple green syn-
0 mg/mL
thetic approach for the preparation of highly fluorescent CDs by using
10 mg/mL S. officinarum juice as carbon source. The synthesized CDs exhibited an
20 mg/mL average size ~3 nm with intense bright blue color emission under UV-
OD

40 mg/mL light, and these are characterized by UV-visible, fluorescence, FT-IR,

100 mg/mL
and HR-TEM techniques. The synthesized CDs showed high fluores-
200 mg/mL
cence ability for in vitro cellular imaging of bacteria and yeast. As a re-
400 mg/mL
sult, CDs were successfully conjugated with cells of bacteria and yeast
and showed different fluorescence colors (blue, green, and red) de-
pending on the excitation wavelength. Therefore, the present method
allows upward scalability in terms of one-pot green synthesis of fluores-
Time (h)
cent and biocompatible CDs, which can be used as fluorescent imaging
Fig. 8. Cytotoxicity of CDs on (a) Escherichia coli and (b) Saccharomyces cerevisiae. The probes for in vitro and in vivo bioimaging applications in cell biology
values are plotted as mean ± SD (n = 3). and other sensing applications.
 V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27
Acknowledgements
Authors thanks Prof. Z. V. P. Murthy, Chemical Engineering Depart-
ment, S. V. National Institute of Technology, Surat, for providing DLS in-
strument facility to this work. We also thank the Department of Science
and Technology for providing Maya Pro 2000 spectrophotometer
(Ocean Optics, USA) under the Fast-Track Young Scientist Scheme
(2011–2014).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.msec.2014.01.038.
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