Sensors and Actuators B 241 (2017) 190–198

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Sensors and Actuators B: Chemical

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

Green synthesis of carbon dots from rose-heart radish and application

for Fe3+ detection and cell imaging
Wen Liu a,b,∗ , Haipeng Diao b , Honghong Chang a , Haojiang Wang b , Tingting Li b ,
Wenlong Wei a,∗
a
Department of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China
b
School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, China

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

Article history: Herein, a simple, green, and low-cost way was developed in the synthesis of fluorescent carbon dots (CDs)
Received 15 June 2016 with well-distributed size, using one-pot hydrothermal treatment of rose-heart radish. The as-prepared
Received in revised form 13 October 2016 carbon dots exhibit exceptional advantages including high fluorescent quantum yield (13.6%), excellent
Accepted 14 October 2016
biocompatibility, low-toxicity, and satisfactory chemical stability. More strikingly, as-synthesized N-CDs
Available online 18 October 2016
generate strong response to Fe3+ ions and gives rise to the fluorescence quenching. This phenomenon
was used to develop a fluorescent method for facile detection of Fe3+ with a linear range from 0.02
Keywords:
to 40 ␮M and a detection limit of 0.13 ␮M (S/N = 3), and further extended to measure environmental
Carbon dots
Fluorescent
water samples with satisfactory recoveries. Eventually, the low toxicity and strongly fluorescent carbon
Fe3+ sensing dots were applied for cell imaging and the quenched fluorescence by adding Fe3+ , demonstrating their
Cell multicolor imaging potential towards diverse applications.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
As a new kind of nanomaterials in the nanocarbon family, fluo-
rescent carbon nanoparticles have drawn tremendous attention in
recent research because of their unique optical and chemical prop-
erties. Unlike traditional organic dyes and semiconductor quantum
dots, fluorescent carbon nanoparticles show distinguished proper-
ties such as stable photoluminescence, good biocompatibility and
low toxicity [1–3]. This kind of material has attracted tremendous
attention in the fields of biological labeling, photocatalysis [4], sens-
ing [5] and biomedicine [6]. Carbon dots (CDs) are generally defined
as carbon nanomaterials with sizes below 10 nm and considered to
consist of an amorphous or crystalline core with predominant sp2
carbon and an oxidized carbon surface with carboxyl group [7].
Recently, nitrogen-doped CDs (N-CDs) have been synthesized
to improve the QY of CDs because nitrogen atoms having a compa-
rable atomic size and five valence electrons available for bonding
with carbon atoms [8]. Moreover, nitrogen doping can effectively
tune the intrinsic properties of CDs, including their electronic prop-
erties, surface and local chemical reactivity [9–11]. In terms of using
∗ Corresponding authors at: Department of Chemistry and Chemical Engineering,
Taiyuan University of Technology, Taiyuan 030024, China.
E-mail addresses: liuwen@sxmu.edu.cn (W. Liu), weiwenlong@tyut.edu.cn
(W. Wei).
a nitrogen source from inorganic molecules, for example, the N-CDs
can be obtained by mixing the glucose and aqueous ammonia for
14 months [12]. However, all these processes are time-consuming
and not environmentally friendly, which restricts their wider appli-
cation. Recently, some natural products containing nitrogen, such
as papaya [13], winter melon [14] and konjac flour [15] were used
as the precursors to prepare N-CDs by hydrothermal treatment.
Unfortunately, the N-CDs synthesized from these cheap and abun-
dant precursors did not achieve high QY values (usually <10%),
causing difficulty for practical application. Wei et al. [16] developed
an economical, and green preparative strategy toward photolu-
minescent (PL) CDs with a quantum yield (QY) of approximately
7.7% by hydrothermal process using cornflour as a carbon source.
Sun et al. [17] demonstrated the use of flour as carbon source
for microwave-assisted rapid green synthesis of CDs with a QY of
approximately 5.4%. Zou et al. [18] described a facile approach for
the synthesis of fluorescent CDs using watermelon peel as carbon
source with a QY of 7.1%. Therefore, there is still space to search for
a low-cost precursor serving as both a carbon source and a nitrogen
source, and to develop a green, simple and rapid synthesis of highly
photoluminescent N-CDs on a large scale. Most natural materials
possess complex component, CDs made from these materials usual
possess various surface groups and special performance.
Rose-heart radish was often regarded as healthiest food as it
contains anthocyanin, which is a kind of water soluble pigment

http://dx.doi.org/10.1016/j.snb.2016.10.068
0925-4005/© 2016 Elsevier B.V. All rights reserved.
 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198
and protect human beings from cardiovascular diseases. For abun-
dant carbon, nitrogen and oxygen elements from crude protein,
amino acid, carbohydrates and vitamin in rose-heart radish, so as-
prepared N-CDs fabricated from rose-heart radish is easy to be
functionalized. In addition, rose-heart radish is quite common and
low cost in everyday life, which is quite suitable for large-scale
fabrication of N-CDs.
Meanwhile, as the increasing amount of Fe3+ in environmen-
tal water became great threats for human health, the needs to
develop sensitive, selective and inexpensive sensor for Fe3+ detec-
tion aroused. In this contribution, we have developed an extreme
green and economical strategy for the fabrication of high lumi-
nescent N-CDs by one-pot hydrothermal treatment of rose-heart
radish. The as-synthesized N-CDs show high fluorescent with QY of
13.6% and excellent selectivity and sensitivity to Fe3+ . The detection
limit for Fe3+ could achieve as low as 0.13 ␮M (S/N = 3), which made
this material a suitable candidate for environmental Fe3+ detection.
The results from the measurement of Fe3+ in river water verified our
assumption and manifested their potential applications in practi-
cal applications. Meanwhile, the carbon dots were employed for
cell imaging, demonstrating their potential in biomedical fields.
2. Experimental
2.1. Materials
AgNO3 , AlCl3 , CaCl2 , CdCl2 , CrCl3 , CoCl2 , CuCl2 , FeCl2 , FeCl3 ,
Hg(NO3 )2 , KCl, MgCl2 , MnCl2 , NaCl, NiCl2 , PbCl2 , ZnCl2 , NaH2 PO4
and Na2 HPO4 were purchased from Shanghai Aladdin Reagent
Co., Ltd. (Shanghai, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) was obtained from Solarbio
(Beijing, China). The rose-heart radish was purchased from local
supermarket (Taiyuan, China). All chemicals were of analytical
grade and used as received without further purification. The deion-
ized water was used throughout for all experiments.
2.2. Instruments
The transmission electron microscopic (TEM) and high-
resolution transmission electron microscopic (HRTEM) images
were taken using a TECNAI G2 F20 microscope (FEI, America) at
200 KV. The samples for TEM characterization were prepared by
coating the given volume of N-CDs dispersion on a carbon-coated
copper grid, and then dried under vacuum at 50 ◦ C for 12 h. The size
distribution of N-CDs was performed by counting over 100 parti-
cles. Fluorescent photographs of N-CDs under UV light of 365 nm
were operated with ZF-6 ultraviolet analyzer from Shanghai Jia-
peng electronic instrument factory. Meanwhile, UV/Vis absorption
spectra were recorded by a Mapada UV-6100 spectrophotometer
(Shanghai, China). Fourier Transform Infrared Spectroscopy (FTIR)
spectra were recorded by Varian FTIR-640 spectrometer (Varian,
America), the sample with 1 mg diluted by KBr (ratio1:200) was
pressed into the disk. Elemental and functional groups analysis
were obtained by ESCALAB 250 X-ray photoelectron spectrometer
from Thermo Scientific Company using 300 W Al K␣ radiation. All
fluorescence measurements were performed on a Cary Eclipse fluo-
rescence spectrophotometer (Varian, America) with excitation slit
set at 5 nm band pass and emission at 5 nm band pass in 1cm × 1 cm
quartz cell.
2.3. Preparation of N-CDs
N-CDs were synthesized via a hydrothermal method using rose-
heart radish as carbon precursor. In a typical synthesis, 2.0 g fresh
chopped rose-heart radish was added into 10 mL ultrapure water
and stirred for 30 min. The mixture was then transferred into a
191
25 mL Teflon-lined autoclave and heated at 180 ◦ C for 3 h in an
oven. Ultimately, the suspension was subjected to centrifugation
(13000 rpm, 15 min) and passed through a 0.22 ␮m micron filter
and dialysis against deionized water though a dialysis membrane
(500–1000 MWCO) for 24 h.
2.4. Measurement of fluorescence QY
The QY of N-CDs was determined according to an established
procedure. In brief, quinine sulfate in 0.1 M H2 SO4 (QY: 0.54 at
340 nm) was chose as the standard. In order to minimize the re-
absorption effects, absorbencies in the 10 mm fluorescence cuvette
were kept under 0.05 at the excitation wavelength (360 nm). The
QY of the CDs was calculated by the following equation:
I AR n2
Q = QR × × × 2 (1)
IR A nR
Where Q is the QY, I is the measured integrated fluorescent emission
intensity, n is the refractive index of the solvent, and A refers to the
absorbance. The subscript R refers to the corresponding parameter
of known fluorescent standard.
2.5. Fluorescence assay of Fe3+
The detection of Fe3+ was performed at room temperature in
10 mM phosphate buffered saline (PBS, pH = 7.4). In a typical assay,
20 ␮L of N-CDs dispersion (0.2 mg/mL) was added into 1000 ␮L of
PBS, followed by the addition of Fe3+ standard with various con-
centrations. The fluorescence emission spectra were recorded after
reaction for 3 min at room temperature. The sensitivity and selec-
tivity measurements were conducted in triplicate. The selectivity
of Fe3+ sensing was confirmed by adding other common metal ions
stock solutions (including Ag+ , Al3+ , Ca2+ , Cd2+ , Cr3+ , Co2+ , Cu2+ ,
Fe2+ , Hg2+ , K+ , Mg2+ , Mn2+ , Na+ , Ni2+ , Pb2+ and Zn2+ ions) instead
of Fe3+ and determining in the same way. The fluorescence was
recorded at the excitation wavelength of 330 nm. All experiments
were performed at room temperature.
To determine Fe3+ detection function of prepared N-CDs sen-
sor in environmental waters, water samples collected from the Fen
river (Taiyuan, Shanxi, China) were utilized to replace ultrapure
water. Water samples were filtered through a 0.22 ␮m membrane
prior to detection. Aliquots (1000 ␮L) of this sample solution were
spiked with standard iron ions solutions (final concentration in the
range of 2 ␮M–40 ␮M). The spiked samples were then diluted to
2 mL with PBS containing N-CDs and then analyzed by the above
described method.
2.6. MTT assay
Cell viability was measured by using a 3-(4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Briefly,
Human uterine cervical squamous cell carcinoma (SiHa) cells were
seeded in 96-well culture plates at a density of 1 × 104 cells per well
in Dulbecco’s modified eagle medium (DMEM) containing 10% calf
serum in a 5% CO2 environment and allowed to grow over 12 h (the
cells reached 70–80% confluence). Cells without treatment with N-
CDs were taken as a control. MTT solution (20 ␮L, 5 mg/mL) was
added to each well and the cells were incubated for another 4 h, fol-
lowed by removing the culture medium with MTT, and then 150 ␮L
of DMSO was added. The final mixture was shaken for 10 min at
room temperature. The absorbance of MTT at 490 nm was exam-
ined by micro-plate reader (ELx808, Biotek). Cell viabilities were
presented as the percentage of the absorbance of N-CDs treated
cells to the absorbance of non-treated cells and plotted as N-CDs
concentration. The experiments were repeated three times.
192 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198

2.7. Cells culture and imaging

Human uterine cervical squamous cell carcinoma (SiHa) cells

were cultured in DMEM medium containing 10% FBS routinely
under a humidified atmosphere containing 5% CO2 , and then har-
vested for subculture using trypsin (0.05%, Gibco/Invitrogen) at
37 ◦ C. SiHa cells were subculture onto a 35 mm × 35 mm Petri dish
with a glass bottom, and then allowed to grow for 24 h for attach-
ment. The cells were cultured with 0.5 mg/mL N-CDs and 0.5 mg/mL
N-CDs/100 ␮M Fe3+ , respectively. After incubation for 3 h, the SiHa
cells were harvested using 0.25% trypsin/0.020% EDTA, washed Fig. 1. Diagram for the synthesis of N-CDs from rose-heart radish, along with pho-
three times (1.0 mL each) with pH 7.4 PBS, and kept in PBS for tograph of the corresponding sample under 365 nm UV lamp excitation.
optical imaging by an Olympus FV1000 laser scanning confocal
microscope (CLSM) with 10× and 40× objective (Tokyo, Japan). In
N-CDs were spherical and well dispersed. In addition, Fig. 2 B
addition, N-CDs-stained SiHa cells were also treated with Fe3+
showed that the lattice spacing is ca. 0.226 nm, observed using
(100 ␮M) in 2 mL PBS (pH 7.4) for dynamic fluorescence imaging
high-resolution TEM, corresponded to that of graphitic carbon, rep-
for additional 10 min.
resenting the graphitic nature of the N-CDs. Fig. 2C demonstrated
that the size distribution of the N-CDs was relatively small and
3. Results and discussion the majority falls within the range from 1.2 to 6.0 nm. The cor-
responding nanoparticle size distribution histogram was obtained
3.1. Synthesis and characterization of N-CDs by counting one hundred particles and the average diameter of the
N-CDs was around 3.6 nm. Carbon dots are considered to consist
As shown in Fig. 1, The N-CDs were synthesized using rose-heart of an amorphous or crystalline core with predominant sp2 carbon
radish as carbon precursor via a simple hydrothermal treatment at and an oxidized carbon surface with carboxyl and hydroxyl groups.
180 ◦ C for 3 h. The size distribution and morphology of the obtained To identify the functional groups of N-CDs, the FTIR spectrum was
N-CDs were characterized by TEM (Fig. 2A). As shown in Fig. 2A, given in Fig. 2D. The broad band in the region of 3021–3680 cm−1

Fig. 2. (A) TEM image of N-CDs, scale bar is 10 nm. (B) HRTEM image of N-CDs, scale bar is 2 nm. (C) Diameter distribution of N-CDs. (D) FTIR spectrum of N-CDs (The inset
displays the diagram for the chemical functional groups of N-CDs.).
 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198
Fig. 3. (A) XPS spectrum of N-CDs. (B)–(D): High-resolution C1s, O1 s and N1 s peaks of the N-CDs, respectively.
arouse from stretching vibrations of O H and N H, the small band
at 2930 cm−1 could be identified as the stretching vibrations of
C H bonds. The peaks at 1648 and 1409 cm−1 indicated to the exis-
tence of COO− [19]. The band at 1531 cm−1 was attributed to the
stretching vibrations of C C. The broad band around 1133 cm−1
was ascribed to the bending vibrations of C O. The inset of Fig. 2D
displays diagram for the chemical functional groups of as-prepared
N-CDs.
The surface functional groups and element states of N-CDs were
also characterized by the XPS. As shown in Fig. 3A, the XPS spectrum
of C-dots displayed three typical strong peaks at 285.2, 400.1, and
531.5 eV, which were attributed to C1s, N1 s and O1s, respectively.
The XPS analysis of the elemental composition of N-CDs demon-
strated that these CDs were mainly composed of carbon (64.14%),
nitrogen (8.33%) and oxygen (27.53%). The amount of N (8.33%)
was higher than that of carbon particles in the literature (6.88%,
4.23%) [20,21], which was attributed to self-surface passivation of
carbon particles. The high resolution spectrum of C1s in Fig. 3B
exhibited four main peaks. The binding energy peak at 284.5 eV
was attributed to C C bond, The peak at about 285.6 eV could be
assigned to C N, while the peak around 286.5 eV could be assigned
to C OH, and the peak around 288.1 eV could be ascribed to C O
[21–23]. The high resolution spectrum of O1 s (Fig. 3C) displays
two peaks at 531.2 and 532.3 eV, which can be attributed to C O
and C OH/C O C groups, respectively [21,24]. The N1 s spectrum
(Fig. 3D) could be divided into two peaks. The main peak with the
binding energy of 399.9 eV was attributed to the C N C. Another
small peak at 401.2 eV originated from N H [25].
The functional groups identified by the XPS were in good agree-
ment with FTIR. These XPS and FTIR data demonstrated that the
as-prepared N-CDs contained functional groups with oxygen and
nitrogen elements, which endowed this fluorescent material excel-
lent water solubility and facilitated its further modifications and
applications.
193
3.2. Optical properties
The optical properties of N-CDs were explored in Fig. 4. As shown
in Fig. 4A, the UV–vis absorption spectra of the N-CDs dispersed in
water showed a strong absorption peak at 281.5 nm corresponding
to the n-␲* transition of C O bond [26,27], which was similar to the
CDs made from grass [21]. The emission spectra in Fig. 4A showed
that a strong emission with the maximum emission wavelength at
420 nm could be observed under excitation at 330 nm. Very bright
blue luminescence was seen under the illumination of UV (365 nm)
light in aqueous solution (as shown in the inset of Fig. 4A). The QY of
the C-dots was calculated to be about 13.6% with quinine sulfate as
standard, demonstrating the good fluorescence properties [28,29].
This value is higher than those of CDs reported previously
[16,20,30–33]. Similar to the common CDs, the prepared C-dots
exhibited excitation-dependent emission character. As shown in
Fig. 4B, the fluorescent peak red-shifted with the increase of exci-
tation wavelength from 280 nm to 500 nm, and the strongest
emission spectrum was obtained at the excitation wavelength of
330 nm. The inset of Fig. 4B displays corresponding normalized
spectra. These behaviors resulted from the different sizes of nano-
CDs and a distribution of different surface states due to the different
organic groups on CDs’ surface [34].
Then the stability of the fluorescent CDs was studied. As shown
in Fig. S1(A), the effect of ionic strength (in terms of the concentra-
tion of KCl) on the PL stability of N-CDs was investigated. The PL
intensity and spectral feature of N-CDs did not change much under
different concentrations of KCl which was beneficial since it was
necessary for N-CDs to be used in the presence of various salt con-
centrations in practical applications. In Fig. S1(B), the effect of pH
on the fluorescence behavior of the N-CDs was also explored. The
fluorescence was enhanced with the increase of pH values from
2.0 to 7.0. However, when pH was higher than 8.0, the fluorescent
intensity decreased dramatically with the increase of pH values.
194 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198

Fig. 4. (A) Absorbance, excitation and emission spectra of the N-CDs. (The insets display the images of N-CDs under daylight and UV irradiation.) (B) Excitation dependent
emission spectra of the N-CDs. (The inset displays corresponding normalized spectra.). (For interpretation of the references to colour in the text, the reader is referred to the
web version of this article.)

Since the PL intensity was highest and maintains fairly constant that the N-CDs had excellent stability, making them promising for
at the physiological pH, it had potential for application in cellular meaningful applications and commercial purpose.
imaging. The phenomena were similar to those of carbon nanocrys-
tals obtained by electrooxidation of graphite, suggesting the CDs
3.3. Sensing of Fe3+
share the same luminescent origin as the carbon nanocrystals [35].
In addition, the fluorescence intensity did not display significant
The PL quenching effect of various cations on N-CDs was investi-
change after continuous irradiation under a 500 W Xe lamp for sev-
gated. Different metal ions including Ag+ , Al3+ , Ca2+ , Cd2+ , Cr3+ , Co2+ ,
eral hours (Fig. S1(C)), indicating the excellent photo-stability of
Cu2+ , Fe2+ , Hg2+ , K+ , Mg2+ , Mn2+ , Na+ , Ni2+ , Pb2+ and Zn2+ and Fe3+ of
the fluorescent probe. Similar observation was obtained when the
each at a concentration of 300 ␮M were reacted with a N-CDs solu-
sample was stored for 3 months (Fig. S1(D)). These results indi-
tion (0.20 mg/mL). As shown in Fig. 5A, it could be seen that a much
cated excellent photo-stability of the N-CDs. The results indicated
lower fluorescence was observed for N-CDs upon addition of Fe3+
ions while other metal ions showed either no or slight change in

Fig. 5. (A) Selectivity of N-CDs (0.2 mg/mL) solution for Fe3+ over other ionic species. The black bars represent the addition of metal ions (300 ␮M for all the metal ions). The
red bars represent the subsequent addition of 300 ␮M Fe3+ to the N-CDs solution. (B) Sensing principle of the N-CDs based probe for Fe3+ . (C) The fluorescence responses of
the N-CDs (0.2 mg/mL) in PBS solution (pH 7.4) after the addition of different concentrations of Fe3+ . (The inset shows the photos of the corresponding solutions illuminated
under UV light of 365 nm). (D) The Stern-Volmer plot of N-CDs at various concentrations of Fe3+ (0.02–40 ␮M) where F0 /F are the PL intensities of N-CDs (0.2 mg/mL) in the
absence and presence of Fe3+ . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198 195

Fig. 6. Laser scanning confocal microscopy images of SiHa cells incubated with (A) 0.50 mg/mL N-CDs, (B) 0.50 mg/mL N-CDs and 100 ␮M Fe3+ at 37 ◦ C for 3 h. The first
left panels of the (a) lines show the bright-field images of SiHa cells. The second, third and fourth panels of the (a) lines are cell images taken at ␭ex /␭em of 405/450 ± 25,
488/520 ± 25 and 543/650 ± 25 nm, respectively. The first panels of the (b) lines are the merged images of the first and second panels of the (a) lines. The second panels of
the (b) lines are the merged images of the second and third panels of the (a) lines. The third panels of the (b) lines are the merged images of the second and fourth panels of
the (a) lines. The fourth panels of the (b) lines are the merged images of the third and fourth panels of the (a) lines. (For interpretation of the references to colour in the text,
the reader is referred to the web version of this article.)

the PL intensity relative to the free N-CDs. This result revealed that
the as-synthesized N-CDs were highly selective toward Fe3+ ions
over the other metal ions and could be developed as an efficient
fluorescence sensor for Fe3+ ions. Among these metal ions, Fe3+ dis-
played the strongest PL quenching effect on N-CDs. Furthermore,
no obvious interference was found even with high concentrations
(up to 300 ␮M) of other metal ions. The possible mechanism was
shown in Fig. 5B. Such a specific fluorescence quenching effect may
originated from the strong interactions between Fe3+ ions and the
surface groups of N-CDs which transfered the photoelectrons from
N-CDs to Fe3+ ions [36,37]. Fe3+ was known to be able to absorb on
the surface of N-CDs and coordinate with phenolic hydroxyl group
on edge of carbon dots. Due to this coordination interaction, elec-
tron in the excited state of N-CDs would transfer to the unfilled
orbit of Fe3+ , and lead to nonradiative electron/hole recombina-
tion, which results in the fluorescence quenching. Fig. 5C showed
the fluorescence quenching of N-CDs at various concentrations of
Fe3+ ions. With increasing Fe3+ ions concentration, the fluores-
cence intensity of N-CDs decreased progressively, revealing that
the sensing system is sensitive to Fe3+ concentration. The inset of
Fig. 5C displays the visual fluorescence change of an N-CDs solution
(0.20 mg/mL) by Fe3+ (300 ␮M) under UV light of 365 nm. It was
very obvious that the blue emission disappeared after adding Fe3+
to the N-CD solution. Fe3+ ions are indispensable for a large number
of living systems and play an important role in many biochemical
processes. The detection of Fe3+ ions through a visible fluorescent
method would be of considerable benefit. Fig. 5D displayed the
Stern-Volmer plot of N-CDs with increasing concentration of Fe3+
(F0 /F against concentration of Fe3+ ), where F0 and F were the PL
intensities of N-CDs at ␭ex /␭em of 330/420 nm in the absence and
presence of Fe3+ , respectively. The quenching efficiency was fitted
by the Stern-Volmer equation:
F0
= 1 + Ksv [Q ] (2)
F
Where Ksv is the Stern-Volmer quenching constant and [Q] is the
Fe3+ concentration. The correlation coefficient (R2 ) was 0.9927
196 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198

Fig. 7. Dynamic fluorescence imaging of SiHa cells cultured in 0.50 mg/mL N-CDs at 37 ◦ C for 3 h and subjected to 100 ␮M Fe3+ for (A) 0.0, (B) 1.0 and (C) 10 min. The first,
second and third panels are cell images taken at ␭ex /␭em of 405/450 ± 25, 488/520 ± 25 and 543/650 ± 25 nm, respectively.

over the concentration range of 0.02–40 ␮M, while the plot did Table 1
Recoveries of Fe3+ ions in Fen River water detected by the proposed method (n = 3).
not fit a linear equation over the whole concentration range of
0.02–300 ␮M, indicating that both dynamic and static quenching Sample Added (␮M) Measured (␮M) Recovery (%) RSD (%)
processes occur in this sensor system [38]. The F0 /F curve was lin- Fen River water 0 0.6 – 1.12
early related to the concentration of Fe3+ in the range 0.02–40 ␮M, 2 2.46 93.00 1.25
indicating their excellent sensing properties in the detection of 4 4.75 103.75 2.23
trace Fe3+ . The limit of detection (LOD) was calculated by taking the 10 10.87 102.70 0.98
20 20.55 99.75 1.45
PL intensity equal to 3 times the standard deviation of the intensity
40 40.88 100.70 1.98
at the blank (n = 11) divided by the slope of the calibration graph.
The LOD of the proposed sensor was determined as 0.13 ␮M. The
LOD is lower than some other reported values [39–42].
responses were recorded. The recoveries were calculated by the
following equation:
3.4. Detection of Fe3+ in environmental samples
Recovery = [(C 2 −C 1 )/C 0 ] × 100% (3)
To test its practicality, the developed method was extended
to determine the concentration of Fe3+ in environmental water Where C0 is the concentration of Fe3+ added into the real sam-
samples from the Fen river in our city. All the samples were fil- ples; C1 and C2 is the concentration of Fe3+ in real samples before
tered through a 0.22 ␮m membrane, followed by centrifugation at and after adding the standard Fe3+ . The results had been illustrated
15000 rpm for 15 min to remove any suspended particles. The flu- in Table 1. The recoveries of all samples were between 93.00 and
orescence intensity of the N-CDs decreased when the samples had 103.75% and the low relative standard deviations (RSDs), range
been spiked with standard solutions. The concentration of Fe3+ in from 0.98% to 2.23%, demonstrating the proposed method has good
the real samples was determined by the proposed method. Then accuracy. These results demonstrate that it is feasible to detect
different concentration of Fe3+ was added and the fluorescence Fe3+ in environmental waters with the present method. In addi-
 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198
tion, the simple, low-cost, green preparative strategy would favour
the application and development.
3.5. Cell cytotoxicity assay
Herein, to explore the potential application of N-CDs in living
cell, it was crucial to assess the toxicity of N-CDs to the human uter-
ine cervical squamous cell carcinoma (SiHa) by the MTT assay. In
the MTT assay, MTT could be reduced by the active cellular enzymes
in the cells to insoluble blue-violet formazan crystals. The quanti-
tative information about the cytotoxicity of CDs would be obtained
by the measurement of absorption on the cells. We performed the
MTT assay in SiHa cells with concentrations from 0.0 mg/mL to
1.0 mg/mL for 24 h. Fig. S2 depicts the cell viability studies under
various concentrations of N-CDs. The survival rates are higher than
85% even under a high concentration (1.0 mg/mL) of N-CDs. These
results demonstrate that N-CDs display low toxicity to the cells,
which makes them a potential candidate for bioimaging of live cells
or other biomedical applications.
3.6. Application of N-CDs in cell imaging
It has been reported that water-soluble fluorescent CDs are ideal
cell imaging probes with minimal cytotoxicity [42–45]. Herein,
SiHa cells were used to explore the potential of our N-CDs as a
bioimaging agent. The cellular uptake of N-CDs was then observed
by laser scanning confocal microscopic (LSCM) as depicted in Fig. 6.
The bright-field images of the SiHa cells incubated with N-CDs and
N-CDs/Fe3+ (first left panels in Fig. 6) indicate clearly the normal
morphology of the cells, verifying that N-CDs and N-CDs/Fe3+ are
biocompatible and possess minimum toxicity to the cells. As shown
in Fig. 6A, it was obvious that the transfected SiHa cells became
quite bright owing to the strong fluorescence from N-CDs, indi-
cating a large amount of N-CDs have been internalized into the
cells. The cells displayed blue (the second panel in Fig. 6A (a)),
green (the third panel in Fig. 6A (a)), and red (the fourth panel in
Fig. 6A (a)) emissions when they were excited with 405, 488, and
543 nm lasers, respectively. And the every two-channel lumines-
cence images for the same scanning area overlapped well (Fig. 6A
(b)). This multicolor emission showed a great advantage of the N-
CDs over other labeling agents because it gave us much space to
choose the wavelength for observation. In addition, the cells incu-
bated with N-CDs/Fe3+ displayed weak blue (the second panel in
Fig. 6B (a)), green (the third panel in Fig. 6B (a)), and red (the
fourth panel Fig. 6B (a)) emissions when they were excited with
405, 488, and 543 nm lasers, respectively. The cells incubated with
N-CDs/Fe3+ emited weaker than that of N-CDs, attributing to the
fact that Fe3+ could quench N-CDs in aqueous solution. Dynamic flu-
orescence imaging was performed to observe quenching process in
real time by confocal fluorescence microscope. SiHa cells were incu-
bated with N-CDs (0.5 mg/mL) for 3 h at 37 ◦ C, and then with Fe3+
(100 ␮M) for 10 min. Fig. 7 depicted the LSCM images of SiHa cells
incubated with N-CDs and exposed to Fe3+ at various times. The
cell images were taken at Eex /Eem of 405/450 ± 25, 488/520 ± 25
and 543/650 ± 25 nm, respectively. After Fe3+ ions were added, all
the blue, green and red emissions diminished with the increase
in incubation times with Fe3+ in the cells, which were consistent
with the results in Fig. 6. Our results showed that N-CDs could be
used potentially to image Fe3+ in biosystem under confocal fluores-
cence microscope. In other words, as-prepared N-CDs could be an
effective probe for monitoring Fe3+ in biosystem.
4. Conclusions
In summary, we had described a green and low cost synthesis
route to fabricate highly fluorescent N-CDs using rose-heart radish
197
as the precursor and demonstrated their applications in Fe3+ sens-
ing. The N-CDs had outstanding stability such that their PL intensity
did not change even after irradiation by a 500 W Xe lamp for three
hours or storage for three months. The as-prepared N-CDs possess
good biocompatibility and can be used as excellent fluorescence
probes for multicolor imaging. Meanwhile, the obtained CDs had
been utilized as a novel sensing probe for sensitive detection of
Fe3+ ions, and further extended to measure environmental water
samples with satisfactory recovery. Eventually, the low toxicity
and strongly fluorescent carbon dots were applied for cell imag-
ing and the quenched fluorescence by adding Fe3+ , demonstrating
their potential towards diverse applications.
Acknowledgments
This work was financially supported by the Natural Science
Foundation of Shanxi Province (Grant No. 2013011060-3) and
the Youth Foundation of Shanxi Medical University (Grant No.
02201306).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2016.10.068.
References
[1] H.T. Li, Z.H. Kang, Y. Liu, S.T. Lee, Carbon nanodots: synthesis, properties and
applications, J. Mater. Chem. 22 (46) (2012) 24230–24253.
[2] S.N. Baker, G.A. Baker, Luminescent carbon nanodots: emergent nanolights,
Angew. Chem. Int. Ed. 49 (38) (2010) 6726–6744.
[3] P.G. Luo, S. Sahu, S.T. Yang, S.K. Sonkar, J.P. Wang, H.F. Wang, G.E. LeCroy, L.
Cao, Y.P. Sun, Carbon quantum dots for optical bioimaging, J. Mater. Chem. B 1
(16) (2013) 2116–2127.
[4] L. Cao, S. Sahu, P. Anilkumar, C.E. Bunker, J.A. Xu, K.A.S. Fernando, P. Wang,
E.A. Guliants, K.N. Tackett, Y.P. Sun, Carbon nanoparticles as visible-light
photocatalysts for efficient CO2 conversion and beyond, J. Am. Chem. Soc. 133
(13) (2011) 4754–4757.
[5] L. Zhou, Y.H. Lin, Z.Z. Huang, J.S. Ren, X.G. Qu, Carbon nanodots as fluorescence
probes for rapid, sensitive, and label-free detection of Hg2+ and biothiols in
complex matrices, Chem. Commun. 48 (8) (2012) 1147–1149.
[6] Y.F. Wu, H.C. Wu, C.H. Kuan, C.J. Lin, L.W. Wang, C.W. Chang, T.W. Wang,
Multi-functionalized carbon dots as theranostic nanoagent for gene delivery
in lung cancer therapy, Sci. Rep. 6 (2016) 21170–21182.
[7] B.C. Zhu, S.Y. Sun, Y.F. Wang, S. Deng, G.N. Qian, M. Wang, A.G. Hu, Preparation
of carbon nanodots from single chain polymeric nanoparticles and theoretical
investigation of the photoluminescence mechanism, J. Mater. Chem. C 1 (3)
(2013) 580–586.
[8] Y. Li, Y. Zhao, H.H. Cheng, Y. Hu, G.Q. Shi, L.M. Dai, L.T. Qu, Nitrogen-doped
graphene quantum dots with oxygen-rich functional groups, J. Am. Chem.
Soc. 134 (1) (2012) 15–18.
[9] J. Liu, X.L. Liu, H.J. Luo, Y.F. Gao, One-step preparation of nitrogen-doped and
surface-passivated carbon quantum dots with high quantum yield and
excellent optical properties, RSC Adv. 4 (15) (2014) 7648–7654.
[10] Q. Liu, B.D. Guo, Z.Y. Rao, B.H. Zhang, J.R. Gong, Strong two-photon-induced
fluorescence from photostable, biocompatible nitrogen-doped graphene
quantum dots for cellular and deep-tissue imaging, Nano Lett. 13 (6) (2013)
2436–2441.
[11] C.Z. Zhu, J.F. Zhai, S.J. Dong, Bifunctional fluorescent carbon nanodots: green
synthesis via soy milk and application as metal-free electrocatalysts for
oxygen reduction, Chem. Commun. 48 (75) (2012) 9367–9369.
[12] L.B. Tang, R.B. Ji, X.M. Li, K.S. Teng, S.P. Lau, Energy-level structure of
nitrogen-doped graphene quantum dots, J. Mater. Chem. C 1 (32) (2013)
4908–4915.
[13] N. Wang, Y.T. Wang, T.T. Guo, T. Yang, M.L. Chen, J.H. Wang, Green preparation
of carbon dots with papaya as carbon source for effective fluorescent sensing
of Iron(III) and Escherichia coli, Biosens. Bioelectron. 85 (2016) 68–75.
[14] X. Feng, Y.Q. Jiang, J.P. Zhao, M. Miao, S.M. Cao, J.H. Fang, L.Y. Shi, Easy
synthesis of photoluminescent N-doped carbon dots from winter melon for
bio-imaging, RSC Adv. 5 (40) (2015) 31250–31254.
[15] X.Y. Teng, C.G. Ma, C.J. Ge, M.Q. Yan, J.X. Yang, Y. Zhang, P.C. Moraiscd, H. Bi,
Green synthesis of nitrogen-doped carbon dots from konjac flour with off–on
fluorescence by Fe3+ and l-lysine for bioimaging, J. Mater. Chem. B 2 (29)
(2014) 4631–4639.
[16] J.M. Wei, X. Zhang, Y.Z. Sheng, J.M. Shen, P. Huang, S.K. Guo, J.Q. Pan, B.X. Feng,
Dual functional carbon dots derived from cornflour via a simple one-pot
hydrothermal route, Mater. Lett. 123 (2014) 107–111.
198 W. Liu et al. / Sensors and Actuators B 241 (2017) 190–198
[17] X.Y. Qin, W.B. Lu, A.M. Asiri, A.O. Al-Youbi, X.P. Sun, Microwave-assisted rapid
green synthesis of photoluminescent carbon nanodots from flour and their
applications for sensitive and selective detection of mercury(II) ions, Sens.
Actuators B 184 (2013) 156–162.
[18] J.J. Zhou, Z.H. Sheng, H.Y. Han, M.Q. Zou, C.X. Li, Facile synthesis of fluorescent
carbon dots using watermelon peel as a carbon source, Mater. Lett. 66 (1)
(2012) 222–224.
[19] L.H. Shi, X.F. Li, Y.Y. Li, X.P. Wen, J.F. Li, M.M.F. Choic, C. Dong, S.M. Shuang,
Naked oats-derived dual-emission carbon nanodots for ratiometric sensing
and cellular imaging, Sens. Actuators B 210 (2015) 533–541.
[20] H. Huang, J. Lv, D. Zhou, N. Bao, Y. Xu, A. Wang, J. Feng, One-pot green
synthesis of nitrogen-doped carbon nanoparticles as fluorescent probes for
mercury ions, RSC Adv. 3 (2013) 21691–21696.
[21] S. Liu, J. Tian, L. Wang, Y. Zhang, X. Qin, Y. Luo, A.M. Asiri, A.O. Al-Youbi, X. Sun,
Hydrothermal treatment of grass: a low-cost green route to nitrogen-doped,
carbon-rich, photoluminescent polymer nanodots as an effective fluorescent
sensing platform for label-free detection of Cu(II) ions, Adv. Mater. 24 (2012)
2037–2041.
[22] S.L. Hu, A. Trinchi, P. Atkin, I. Cole, Tunable photoluminescence across the
entire visible spectrum from carbon dots excited by white light, Adv. Mater.
54 (10) (2015) 2970–2974.
[23] Z. Yang, M. Xu, Y. Liu, F. He, F. Gao, Y. Su, H. Wei, Y. Zhang, Nitrogen-doped
carbon-rich, highly photoluminescent carbon dots from ammonium citrate,
Nanoscale 6 (3) (2014) 1890–1895.
[24] W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A. Asiri, A.O. Al-Youbi, X. Sun,
Economical, green synthesis of fluorescent carbon nanoparticles and their
useas probes for sensitive and selective detection of mercury(II) ions, Anal.
Chem. 84 (2012) 5351–5357.
[25] S. Liu, J. Tian, L. Wang, Y. Luo, J. Zhai, X. Sun, Preparation of photoluminescent
carbon nitride dots from CCl4 and 1,2-ethylenediamine: a
heat-treatment-based strategy, J. Mater. Chem. 21 (32) (2011) 11726–11729.
[26] G. Eda, Y.-Y. Lin, C. Mattevi, H. Yamaguchi, H.-A. Chen, I.-S. Chen, C.-W. Chen,
M. Chhowalla, Blue photoluminescence from chemically derived graphene
oxide, Adv. Mater. 22 (4) (2010) 505–509.
[27] J.I. Paredes, S. Villar-Rodil, A. Martínez-Alonso, J.M.D. Tascón, Graphene oxide
dispersions in organic solvents, Langmuir 24 (19) (2008) 10560–10564.
[28] Y.Z. Liao, V. Strong, Y. Wang, X.-G. Li, X. Wang, R.B. Kaner, Oligotriphenylene
nanofiber sensors for detection of nitro-based explosives, Adv. Funct. Mater.
22 (4) (2012) 726–735.
[29] R. Purbia, S. Paria, A simple turn on fluorescent sensor for the selective
detection of thiamine using coconut water derived luminescent carbon dots,
Biosens. Bioelectron. 79 (2016) 467–475.
[30] D. Wu, X. Huang, X. Deng, K. Wang, Q. Liu, Preparation of photoluminescent
carbon nanodots by traditional Chinese medicine and application as a probe
for Hg2+ , Anal. Methods 5 (12) (2013) 3023–3027.
[31] G. Gedda, C.Y. Lee, Y.C. Lin, H.F. Wu, Green synthesis of Carbon dots from
prawn shells for highly selective and sensitive detection of copper ions,
Sensor. Actuators B 224 (2016) 396–403.
[32] J.M. Wei, J.M. Shen, X. Zhang, S.K. Guo, J.Q. Pan, X.G. Hou, H.B. Zhang, L. Wang,
B.X. Feng, Simple one-step synthesis of water-soluble fluorescent carbon dots
derived from paper ash, RSC Adv. 3 (32) (2013) 13119–13122.
[33] Y.F. Chen, Y.Y. Wu, B. Weng, B. Wang, C.M. Li, Facile synthesis of nitrogen and
sulfur co-doped carbon dots and application for Fe(III) ions detection and cell
imaging, Sens. Actuators B 223 (2016) 689–696.
[34] X.Y. Xu, R. Ray, Y.L. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens,
Electrophoretic analysis and purification of fluorescent single-Walled carbon
nanotube fragments, J. Am. Chem. Soc. 126 (40) (2004) 12736–12737.
[35] Q. Zhao, Z. Zhang, B. Huang, J. Peng, M. Zhang, D. Pang, Facile preparation of
low cytotoxicity fluorescent carbon nanocrystals by electrooxidation of
graphite, Chem. Commun. 41 (2008) 5116–5118.
[36] S. Zhu, Q. Meng, L. Wang, J. Zhang, Y. Song, H. Jin, K. Zhang, H. Sun, H. Wang, B.
Yang, Highly photoluminescent carbon dots for multicolor patterning sensors,
and bioimaging, Angew. Chem. Int. Ed. 52 (14) (2013) 3953–3957.
[37] W. Li, Z. Zhang, B. Kong, S. Feng, J. Wang, L. Wang, J. Yang, F. Zhang, P. Wu, D.
Zhao, Simple and green synthesis of nitrogen-doped photoluminescent
carbonaceous nanospheres for bioimaging, Angew. Chem. Int. Ed. 52 (31)
(2013) 8151–8155.
[38] S. Sahu, B. Behera, T.K. Maiti, S. Mohapatra, Simple one-step synthesis of
highly luminescent carbon dots from orange juice: application as excellent
bio-imaging agents, Chem. Commun. 48 (70) (2012) 8835–8837.
[39] W. Zhu, J. Zhang, Z. Jiang, W. Wang, X. Liu, High-quality carbon dots:
synthesis, peroxidase-like activity and their application in the detection of
H2 O2 , Ag+ and Fe3+ , RSC Adv. 4 (33) (2014) 17387–17392.
[40] K. Qu, J. Wang, J. Ren, X. Qu, Carbon dots prepared by hydrothermal treatment
of dopamine as an effective fluorescent sensing platform for the label-free
detection of iron(III) ions and dopamine, Chem. Eur. J. 19 (22) (2013)
7243–7249.
[41] M. Yang, M.T. Sun, Z.P. Zhang, S.H. Wang, A novel dansyl-based fluorescent
probe for highly selective detection of ferric ions, Talanta 105 (2013) 34–39.
[42] Y.M. Zhou, H. Zhou, J.L. Zhang, L. Zhang, J.Y. Niu, Fe3+ -selective fluorescent
probe based on aminoantipyrine in aqueous solution, Spectrochim. Acta A 98
(2012) 14–17.
[43] X. Gong, Q. Hu, M.C. Paau, Y. Zhang, S. Shuang, C. Dong, M.M.F. Choi,
Red–green–blue fluorescent hollow carbon nanoparticles isolated from
chromatographic fractions for cellular imaging, Nanoscale 6 (14) (2014)
8162–8170.
[44] L. Shen, L. Zhang, M. Chen, X. Chen, J. Wang, The production of pH-sensitive
photoluminescent carbon nanoparticles by the carbonization of
polyethylenimine and their use for bioimaging, Carbon 55 (2013) 343–349.
[45] B. Han, W. Wang, H. Wu, F. Fang, N. Wang, X. Zhang, S. Xu, Polyethyleneimine
modified fluorescent carbon dots and their application in cell labeling,
Colloids Surf. B 100 (2012) 209–214.
Biographies
Wen Liu, received his BS and MS degrees from the Department of Chemistry at
Datong University in 2007 and Institute of Molecular Science at Shanxi University in
2010, respectively. Thereafter, he joined Shanxi Medical University in 2010. In 2015,
he became a lecturer. He began his doctoral study at Taiyuan University of Technol-
ogy under the supervision of Professor Wenlong Wei. His current research interests
focus on synthesis novel nanomaterials and application in focuses on biosensors and
bioimaging.
Haipeng Diao, received his BS and MS degrees from the School of Chemistry and
Chemical Engineering at Shanxi University in 1999 and the College of Pharmacy
at Shanxi Medical University in 2006, respectively. He joined the Shanxi Medical
University in 1999. In 2011, he became an associate professor. His current research
interests focus on the fluorescence probe in biosensors.
Honghong Chang, received his MS and PhD degrees from the School of Chemistry
and Chemical Engineering at Taiyuan University of Technology in 2004 and 2009. In
2011, he became an associate professor. His current research interests focus on the
organic functional materials and biological applications.
Haojiang Wang, received his BS and MS degrees from the College of Pharmacy at
Shanxi Medical University in 1999 and 2007, respectively. He joined Shanxi Medical
University in 1999. In 2004, he became a lecturer. His current research interests
focus on chemosensor and biosensor.
Tingting Li, received his BS degree from the Department of Chemistry at Taiyuan
Normal University in 2015. Then, she began her graduate study at Shanxi Med-
ical University in 2015. Her current research interests focus on synthesis novel
nanomaterials and application in biosensors.
Wenlong Wei, PhD, received his MS and PhD degrees from the School of Chemistry
and Chemical Engineering at Taiyuan University of Technology in 1988 and 2009,
respectively. He is a professor in School of Chemistry and Chemical Engineering,
Taiyuan University of Technology. His current research interests are the organic
functional materials and novel nanomaterials novel nanomaterials.