Copper-catalyzed three-component synthesis of pyrimidines from amidines and alcohols

Article abstract of DOI:10.1039/c8ob02694g

An efficient copper-catalyzed one-pot three-component reaction of amidines, primary alcohols and secondary alcohols has been developed to synthesize multisubstituted pyrimidines. The significant merits of this method involve high atom efficiency, good fun

Full text of DOI:10.1039/c8ob02694g

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Concentration-dependent multicolor fluorescent
carbon dots for colorimetric and fluorescent
bimodal detections of Fe3+ and L-ascorbic acid†
Cite this: DOI: 10.1039/c8ay02486c
*
Lihong Shi,
Zhipeng Hou, Caihong Zhang, Guomei Zhang, Yan Zhang,
*
Chuan Dong
and Shaomin Shuang
We present a green and facile strategy for fabrication of concentration-dependent multicolor fluorescent
carbon dots (CDs) by using coffee, salvia, and urea as the starting material via a one-step hydrothermal
method. The obtained CDs emit three different colors of fluorescence (i.e. orange, yellow, and blue) with
their concentration reduction. Moreover, yellow fluorescent CDs (Y-CDs) are utilized as a platform for
colorimetric and fluorescent dual-mode detections of Fe3+ and L-ascorbic acid (L-AA) with high
selectivity and sensitivity. The fluorescence of Y-CDs can be effectively quenched by Fe3+ based on
static quenching and subsequently recovered upon the addition of L-AA due to the competitive
complexation of L-AA with Fe3+. Therefore, the as-prepared Y-CDs could serve as a highly efficient
fluorescent probe for “on–off–on” sequential determination of Fe3+ and L-AA. It is more attractive that
Fe3+ and L-AA can also be visualized by this nanosensor via evident color changes under daylight.
Furthermore, the designed nanosensor can be extended to detect intracellular Fe3+ and L-AA with
outstanding biocompatibility and cellular imaging capability, which holds great promise in biomedical
applications.
Received 14th November 2018
Accepted 23rd December 2018
DOI: 10.1039/c8ay02486c
rsc.li/methods
attributed the observed photoluminescence (PL) red shi to the
difference in the nitrogen content of the CDs and their particle
1. Introduction
size.
Regardless of these opinions, an interesting fact has been
As a rising star in the carbon nanomaterial family, carbon dots
(CDs) have generated intense attention by virtue of their high
photostability, superior biocompatibility, low toxicity, and
excellent optical properties.1–5 In particular, the multicolor
emission endows CDs with numerous exciting applications,
such as bioimaging,6–10 chemical sensing,11,12 solid state
lighting,13–15 medical diagnosis,16 and ngerprint detection.17
During the past few years, multicolor uorescent CDs have been
successfully synthesized by tuning various parameters,
including particle size, excitation wavelength, and carbon
source. Bao et al.6 obtained CDs with various colors of uores-
cence through varying their size by ultraltration. Nie et al.7
synthesized excitation-dependent full-color emissions CDs by
changing the reaction conditions of chloroform and diethyl-
amine. The unique emission characteristics were ascribed to
the surface functional groups which efficiently introduce new
energy levels for electron transitions. Jiang et al.18 prepared
three color CDs, i.e. blue, green, and red emissions, by thermal
treatment using ethanol solution of p-phenylenediamine, o-
phenylenediamine, and m-phenylenediamine, respectively, and
discovered that the luminescence of CDs can be tuned by
changing the concentration and accordingly the intermolecular
interaction as well as the surface electron distribution. Meng
et al.19 produced CDs by the reaction of coal pitch powder with
a mixed solution of formic acid and H2O2 without any external
heating and tuned uorescence emission wavelengths from 630
to 400 nm by changing the concentration of CDs. Chen et al.20
used citrate acid and ethanolamine as precursors to synthesize
CDs with different colors in DMF and an aqueous solution as
the concentration changed, which is due to the existence of
multi-emissive centers in CDs from the core state, the edge state
and surface states, and the polarity of the solution. Wang et al.11
used a facile hydrothermal treatment of the mixed solution of
m-phenylenediamine, ethanol, and ammonia to fabricate CDs
with three strongest PL peaks with concentration reduction.
Nevertheless, all these methods use toxic/expensive solvents or
starting materials. Natural materials-derived concentration-
dependent multicolor uorescent CDs are very rare. Therefore,
the development of the green synthesis of concentration-
tunable multicolor emission CDs is still highly desired.
College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
PR China. E-mail: shilihong@sxu.edu.cn; smshuang@sxu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ay02486c
Iron is an essential element of the human body, which can
increase resistance to diseases, regulate tissue respiration,
prevent fatigue, and constitute hemoglobin. Inadequate and
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excessive iron intake can disturb the cellular homeostasis and dialyzed through a dialysis membrane with MWCO of 500–
induce various biological disorders.21,22 Thus, the reliable 1000 Da for 24 h.
detection of Fe3+ has become indispensable from the viewpoint
of human health. Current detection assays include voltamme-
try, spectrophotometry,23,24 atomic absorption spectrometry,25
and inductively coupled plasma mass spectrometry (ICP-MS).26
However, all these methods suffer from some drawbacks like
the requirement of sophisticated instrumentation and tedious
sample preparation procedures, limiting their practical appli-
cations.2 The uorescence-based Fe3+ probe becomes a good
alternative due to its simple operation, high sensitivity, cost
effectiveness, and rapid response. Also, colorimetry is an
important method of detection which allows a direct analysis of
the substrate simply by the naked eye. However, the detection of
Fe3+ based on CDs by both uorescence and colorimetry has not
been reported yet. In addition, L-ascorbic acid (L-AA) is an
important reducer, which has been used for the prevention and
treatment of vitiligo, cancer, and scurvy.27 Hence, the
measurement of the L-AA level in solution and biological
systems is necessary. Although miscellaneous strategies have
been introduced for the quantitative detection of L-AA, many
endeavors are still continuously being made to search for better
strategies to detect the content of L-AA in biological samples.
In this paper, concentration-dependent multicolor uores-
cent CDs have been prepared by hydrothermal treatment of
coffee, salvia, and urea. The as-synthesized CDs show orange,
yellow, and blue uorescence with their concentration reduc-
tion. Yellow uorescent CDs (Y-CDs) are utilized for uorescent
and colorimetric detection of Fe3+ and L-AA sequentially. In
addition, Y-CDs could image the uctuation of Fe3+ and L-AA in
living cells with appreciable changes in uorescence.
2.3 Characterization
The transmission electron microscopy (TEM) study was carried
out using a JEOL JEM-2100 instrument operating at an accel-
erating voltage of 200 kV. Samples for TEM measurements were
prepared by placing a drop of colloidal solution on a carbon-
coated copper grid and then dried at room temperature. The
UV-vis absorption spectrum of CDs was recorded using
a HITACHI U-2910 UV. Fluorescence spectra were recorded with
a Hitachi F-4500 uorescence spectrophotometer. The Fourier
transform infrared (FTIR) spectrum was recorded on a Bruker
tensor 2 spectrometer using a resolution of 4 cmꢀ1. The sample
with 1 mg diluted with KBr (ratio 1 : 200) was pressed into discs.
Atomic force microscope (AFM) images were obtained using an
AFM Bruker MultiMode 8 in the contact mode. The uorescence
lifetime was measured using an Edinburgh FLS920.
2.4 Fluorimetry for the determination of Fe3+ and L-AA
The detection of Fe3+ was performed at room temperature in
Tris–HCl buffer (pH ¼ 7.4). In a typical run, a 0.1 mL CD
dispersion was added to 0.233 mL Tris–HCl buffer at
38.4 mg Lꢀ1 nal concentration, followed by the addition of Fe3+
standard with various concentrations. The uorescence emis-
sion spectra were recorded aer reaction for 1 min at room
temperature.
The effect of L-AA on the uorescence intensity of the Y-CDs/
Fe3+ solution mixture was conducted as follows. The L-AA
standard with various concentrations was added to the Y-CDs
(38.4 mg Lꢀ1)/Fe3+ (400 mM) solution mixture. The uores-
cence emission spectra were recorded aer reaction for 1 min at
room temperature.
2. Experimental
2.1 Materials
2.5 Cellular imaging
AlCl3, BaCl2, BiCl3, CaCl2, CdCl2, CuCl2, FeCl3, HgCl2, KCl,
MgCl2, MnCl2, NaCl, NiCl2, and ZnCl2 were purchased from
Beijing Chemical Corp (Beijing, China). Coffee and salvia were
purchased from a local market (Taiyuan. China). And urea was
purchased from Sigma Aldrich Trade Co., Ltd (Shanghai. China).
Distilled deionized (DDI) water was obtained from a Millipore
Milli-Q-RO4 water purication system with a resistivity higher
than 18 MU cmꢀ1 (Bedford, MA, USA). Dialysis membranes with
a MWCO of 500–1000 Da were purchased from Beijing Solarbio
Science & Technology Co., Ltd. (Beijing, China). Human hepa-
toma cellular carcinoma (SMMC-7721) cells were purchased
from the Taiyuan Armed Police Hospital (Taiyuan, China).
Human hepatoma cellular carcinoma (SMMC-7721) cells were
cultured in Dulbecco's Modied Eagle Medium (DMEM) sup-
ꢁ
plemented with 10% FBS and incubated at 37 C in a 5% CO2
atmosphere. 0.0384 mg CDs were added to 1.0 mL culture
medium at 38.4 mg Lꢀ1 nal concentration. Aer incubation for
24 h, the SMMC-7721 cells were harvested using 0.25% trypsin/
0.020% EDTA, washed three times (1.0 mL each) with pH 7.4
Tris–HCl. Y-CD-stained SMMC-7721 cells were treated with Fe3+
with a concentration of 400 mM for 2 min. Aer that, the cells
were treated with L-AA (57.14 mM) for 3 min.
All uorescence images were collected with a Zeiss LSM 880
confocal laser-scanning microscope. The cell images were taken
at a lex/lem of 405/435–450, 488/492–577 and 514/620–700 nm,
respectively.
2.2 Synthesis of CDs
The CDs were prepared by hydrothermal treatment of coffee,
salvia and urea. 0.5 g coffee, 0.5 g salvia, and 0.2 g urea were
dissolved in 20 mL DDI water. Then the cloudy solution was
poured into a 50 mL hydrothermal reactor vessel, placed in an
3. Results and discussion
3.1 The synthesis of multicolor uorescent CDs
oven, and heated at 220 ꢁC for 5 h. Aer cooling down to room The successful fabrication of concentration-dependent multi-
temperature, the CDs were collected by removing larger parti- color uorescent CDs was carried out by using coffee, salvia,
cles through centrifugation at 4000 rpm for 20 min and then and urea as the starting material via a one-step hydrothermal
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method (Fig. 1). For this one-step approach, the most favorable aromatic structures, amino, and C]C bonds. As the concen-
feature is that the formation of carbon dots and its surface tration decreases, the intermolecular forces pull the CDs closer
passivation can be realized simultaneously.8 We use inexpen- together, and the functional groups on the surface of CDs
sive coffee as well as salvia as the carbon source and urea as combine to reduce the energy gap, which ultimately leads to
a surface passivation agent. CD formation by a hydrothermal a red shi of excitation/emission maxima.28–30 The photographs
method may involve dehydration, polymerization, pyrolysis, of O-, Y-, and B-CDs under daylight and the indicated wave-
and surface passivation processes in a closed system under self- length excitation are shown in the insets of Fig. 2A–C. Taking
generated pressure and at mild temperature.
into account uorescence color and uorescence intensity, we
nally chose Y-CDs for detection and imaging.
3.2 PL properties of the CDs
PL properties of the as-prepared CDs depend on the concen-
tration as well as excitation wavelengths.11 Fig. 2 shows the PL
spectra of CD solution with a concentration of 128, 38.4, and
2.56 mg Lꢀ1. The strongest emission peaks of these three
samples are around 616, 550, and 436 nm and accordingly the
corresponding samples are named O-, Y-, and B-CDs, respec-
tively. The O-CDs exhibit excitation-independent emission
while Y- and B-CDs exhibit excitation-dependent emission.
Particularly, these three strongest PL peaks barely shi when
the concentrations of O-, Y-, and B-CDs are varied in 76.8–128,
38.4–64 and 2.56–12.8 mg Lꢀ1. The concentration-dependent
properties of CDs could be attributed to their interactions
similar to intermolecular forces.20 The surface state of CDs is
a predominant factor for the uorescence emission process.
The energy gap of the surface states, where electrons and holes
recombine to emit uorescence, correlates closely with the
extent of the p-electron system and surface chemistry. The
surface states of CDs are regulated by carbonyl, hydroxy,
3.3 The characterization of the CDs
The size distribution and morphology of CDs were assessed by
TEM. As shown in Fig. 3A–C, CDs are spherical and well
dispersed. Based on the statistical analysis of above three
hundred particles, the mean diameter is about 2.94 ꢂ 0.04 nm
(the inset of Fig. 3A) and about 88% of CDs fall within the range
from 2.25 nm to 4.75 nm. The high resolution TEM (HRTEM)
image (Fig. 3D) shows that CDs possess a crystalline structure
with a lattice spacing of 0.21 nm, being attributed to the (100)
facet of graphite. This result strongly suggests that the synthe-
sized CDs exhibit a graphite-like structure.31
The AFM images (Fig. S1†) show the surface morphology of
the as-prepared CDs. The AFM topography image (Fig. S1A†)
and three-dimensional image (Fig. S1B†) clearly show that CDs
have good dispersivity. Fig. S1C† is a radial height distribution
map along the line in the AFM topography image. As shown in
Fig. S1D,† the height of CDs is from 1.5 nm to 2.3 nm with
a mean height of ca. 1.92 nm. This is consistent with TEM data.
We used FTIR to identify the functional groups of CDs
(Fig. S2A†). In the FTIR spectrum of CDs, the peak at 3194 cmꢀ1
represents the O–H stretching vibrations, the peak at 1658 cmꢀ1
shows the C–O bending vibrations, the peak at 1654 cmꢀ1
corresponds to the C–N stretching vibrations and N–H bending
vibrations, the peaks at 1477, 1383, 756, and 690 cmꢀ1 are
associated with the C–H bending vibrations, and the peaks at
1232 and 1068 cmꢀ1 suggest the existence of the C–O–C
stretching vibrations. Additionally, the PL intensity of CDs
remains almost constant even aer excitation for 50 min
(Fig. S2B†), suggesting that the photostability of CDs is
eminent. The PL intensities stay almost stable when the pH
value was changed from 7.0 to 9.2 (Fig. S2C†), which shows that
the PL intensity of CDs is pH independent.
Fig.
1 Diagram for the fabrication of concentration-dependent
multicolor fluorescent CDs.
Fig. 2 PL spectra of CDs with different concentrations (A) 128 mg Lꢀ1, (B) 38.4 mg Lꢀ1, and (C) 2.56 mg Lꢀ1 at different excitation wavelengths.
Inset: photographs of the corresponding samples under daylight and indicated wavelength excitation.
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the Y-CDs at various concentrations of Fe3+ were performed
(Fig. 4D). The absorption changes constantly with increasing
Fe3+ concentrations, implying that uorescence quenching can
be attributed to static quenching arising from the formation of
a stable non-uorescent complex between CDs and Fe3+.3,22,33 In
addition, the uorescence lifetime is also effective evidence to
distinguish the quenching type. Thus, uorescence emission
decay curves of Y-CDs in the absence and presence of Fe3+ were
measured (Fig. S3†). The lifetimes of Y-CDs and Y-CDs/Fe3+ are
calculated to be 5.27 ns and 5.45 ns (Table S2†), which proves
that the uorescence quenching of Y-CDs in the presence of Fe3+
is a static quenching process on account of the formation of the
Y-CDs/Fe3+ complex. The consequence of the uorescence life-
time is in accord with that of UV-vis absorption spectra.
The colorimetric sensing behavior of Y-CDs was investi-
gated by monitoring color changes upon addition of different
metal ions. The addition of Fe3+ to Y-CD solution results in an
apparent color change from pale yellow to dark-brown
(Fig. 4E), while other metals cause no change in color. The
color change of the solution may also stem from the complex
interaction between CDs and Fe3+
.
Thus, we can also use
34
Fig. 3 TEM images of CDs: (A) scale bar is 100 nm (inset: size distri-
bution of CDs), (B) scale bar is 20 nm, and (C) scale bar is 10 nm. (D)
HRTEM image of CDs, scale bar is 2 nm.
colorimetric methods to identify Fe3+. Fe3+ ions are transition
metals and contain an unlled d orbital. When CDs coordi-
nate with Fe3+, electrons on the CDs are more likely to enter
the unlled d orbital of Fe3+, and charge transfer occurs,
causing gradual deepening of the solution color. Fig. 4F shows
the color change aer adding different concentrations of Fe3+
under daylight. It can be seen that as the concentration of Fe3+
increases, the color of Y-CD solution continually changes from
pale yellow to dark-brown. These results demonstrate that the
Y-CDs are an intuitive and convenient potential naked-eye
3.4 Fe3+ and L-AA sensing of Y-CDs in aqueous solution
The inuence of 14 kinds of metal ions on the PL intensity of Y-
CDs under pH 7.4 conditions was studied, including Cd2+, Cu2+,
Ca2+, Fe3+, Hg2+, Ba2+, Bi3+, Ni2+, Mg2+, Zn2+, Mn2+, K+, Al3+, and
Na+ (Fig. 4A). Among these metal ions, Fe3+ exhibits the largest
quenching of the uorescence of the Y-CDs while other metal
ions do not have a signicant effect on the uorescence inten-
sity of Y-CDs. Moreover, no obvious interference is observed
even with high concentrations (up to 400 mM) of other metal
ions. In comparison with other ions, the high selectivity of Y-
CDs for Fe3+ may be attributed to the faster coordination of
Fe3+ ions with the N containing functional groups in Y-CDs.32
Therefore, the Y-CDs have high selectivity toward Fe3+ over the
other metal ions and could be designed as an efficient “turn-off”
uorescence Fe3+ sensor. Also, we investigated the Fe3+ sensing
capacity of Y-CDs in the range of 0 mM to 400 mM (Fig. 4B). The
uorescence intensity at 548 nm decreases progressively as the
Fe3+ concentration increases. Meanwhile, the photographs of
a Y-CD solution under 470 nm excitation were taken (the inset
of Fig. 4B) which display that the uorescence color of Y-CD
solution changes from yellow to colorless with the increase of
Fe3+ concentration. Fig. 4C presents PL data versus the
concentration of Fe3+. There is a good linearity between the F/F0
and Fe3+ concentration in the range of 0.5–175 mM and 200–400
mM, respectively. The corresponding regression coefficient (R2)
is 0.99704 and 0.98621, respectively. The detection limit of
27.8 nM is obtained based on a signal-to-noise ratio of 3,
showing lower value than those previously reported for the Fe3+
detection (Table S1†). The results show that the sensor is highly
sensitive and has a wide linear range. To further investigate the
PL quenching mechanism, UV-vis absorption experiments on
sensor for Fe3+
.
At the same time, it is exciting that we have found that the
addition of L-AA could recover the uorescence of Y-CDs
quenched by Fe3+. We studied the inuence of various
external substances on the PL intensity of the Y-CDs/Fe3+ under
pH 7.4 conditions, including cations, anions, small molecules,
and amino acids (Fig. S4†). Among these substances, L-AA
exhibits the largest recovery of the uorescence of Y-CDs/Fe3+,
while other substances have a slight effect. Meanwhile, the
interference experiment of Y-CDs/Fe3+ was also implemented
(Fig. S4†), showing that these substances have no effect on the
recovery of uorescence. These results demonstrate that Y-CDs/
Fe3+ has good selectivity and strong anti-interference ability for
L-AA sensing.
Therefore, the Y-CDs/Fe3+ system could be developed as an
“off–on” uorescence probe for detection of L-AA. As illustrated
in the proposed “on–off–on” sensing process (Fig. 5), the colors
of Y-CDs, Y-CDs/Fe3+, and Y-CDs/Fe3+/L-AA solution vary under
irradiation with daylight and 470 nm excitation. The uores-
cence intensity at 548 nm is gradually recovered with increasing
concentrations of L-AA (Fig. 6A) Meanwhile, the photographs of
Y-CDs/Fe3+ solution under 470 nm excitation were taken (the
inset of Fig. 6A). Fig. 6B presents the uorescence data versus
the concentration of L-AA. In a good range of 3.23–22.08 mM
and 33.33–57.14 mM, respectively, the corresponding regres-
sion coefficient (R2) is 0.99645 and 0.99182, respectively. The
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Fig. 4 (A) Selectivity of Y-CD solution for Fe3+ over other cations under pH 7.4 conditions at 470 nm excitation. The red bars represent the
addition of metal ions (400 mM for all the metal ions) to Y-CD solution. The blue bars represent the subsequent addition of 400 mM Fe3+ to Y-CD
solution. (F0 and F are the fluorescence intensity of Y-CDs in the absence and presence of external ions, respectively). (B) Representative
fluorescence emission spectra of Y-CDs in the presence of various concentrations of Fe3+ (0–400 mM). (C) The relationship between the F/F0 and
Fe3+ concentration from 0 mM to 400 mM. The linear relationship between the fluorescence intensity and Fe3+ concentration in the ranges of
0.5–175 mM and 200–400 mM. (D) UV-vis absorption spectra of Y-CDs in the presence of various concentrations of Fe3+. (E) The color changes of
Y-CD solution to different cations (400 mM) under daylight. (F) The color changes of Y-CD solution to various concentrations of Fe3+ (0–400 mM)
under daylight.
detection limit was calculated to be 1.8 mM for L-AA based on
a signal-to-noise ratio of 3. Compared with previous work
(Table S3†), the proposed L-AA probe is more selective and less
sensitive. The uorescence recovery of Y-CDs/Fe3+ by L-AA is
likely due to the following reasons. First, the uorescence
recovery of Y-CDs/Fe3+ solution is actually attributable to the
stronger binding affinity of L-AA towards Fe3+, which removes
Fe3+ from the surface of Y-CDs. Second, for the Y-CDs/Fe3+
system, Fe3+ oxidizes L-AA to diketogulonic acid and is reduced
to Fe2+ simultaneously. The complex of diketogulonic acid with
Fe2+ has strong absorption peak at 269 nm (Fig. 6C). At the same
time, the color of the system changed from dark-brown to pale
yellow under daylight with the increase of L-AA concentrations
Fig. 5 Photographs of Y-CDs in the absence of Fe3+ and L-AA (first), in
(Fig. 6D). These results also demonstrate that Y-CDs/Fe3+ is
the presence of Fe3+ (second), and in the presence of Fe3+ and L-AA
a potential naked-eye sensor for L-AA.
(third) under daylight and 470 nm excitation.
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Fig. 6 (A) Representative fluorescence emission spectra of Y-CDs/Fe3+ in the presence of various concentrations of L-AA (0–57.14 mM). (B) The
relationship between the F/F0 and L-AA concentration from 0 to 57.14 mM. The linear relationship between the fluorescence intensity and L-AA
concentration in the ranges of 3.23–22.08 mM and 33.33–57.14 mM. (F0 is the fluorescence intensity of Y-CDs in the absence of Fe3+ and L-AA
and F is the fluorescence intensity of Y-CDs/Fe3+ in the presence of L-AA.) (C) UV-vis absorption spectra of Y-CDs/Fe3+ in the presence of various
concentrations of L-AA. (D) The color changes of Y-CDs/Fe3+ solution to various concentrations of L-AA (0–57.14 mM) under daylight.
10 mg Lꢀ1 to 50 mg Lꢀ1. The results demonstrate that more than
3.5 Cell cytotoxicity assay
86.5% of the cells are viable (Fig. S5†), which implies the low
To explore the potential application of CDs in live cell imaging,
it is required that the selected uorescent marker possesses not
toxicity of Y-CDs to cultured cells under the experimental
conditions at a concentration of 38.4 mg Lꢀ1. The Y-CDs were
only optical merits but also low cytotoxicity.35 To evaluate the
internalized into the cells possibly by an endocytosis
cytotoxicity of the as-prepared CDs, we performed the MTT
assay in SMMC-7721 cells with concentrations ranging from
mechanism.36
Fig. 7 (A) Dynamic LSCM fluorescence imaging of SMMC-7721 cells with Y-CDs/Fe3+ and Y-CDs/Fe3+/L-AA (lex/lem: 488 nm/492–577 nm). (B)
LSCM images of SMMC-7721 cells incubated with Y-CDs, Y-CDs/Fe3+ (400 mM), and Y-CDs/Fe3+ (400 mM)/L-AA (57.14 mM). The first, second,
and third panels are cell images taken at a lex/lem of 405 nm/435–450 nm, 488 nm/492–577 nm, and 514 nm/600–700 nm, respectively. The
fourth panel shows the bright field images. The fifth panel is the merged image of the first, second, third and fourth panels. (C) Intracellular
fluorescence responses of Y-CDs, Y-CDs/Fe3+ (400 mM), and Y-CDs/Fe3+ (400 mM)/L-AA (57.14 mM). Data are expressed as mean ꢂ standard
deviation. The intensities of 10 cells are measured. Scale bar is 50 mm.
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3.6 In vitro cellular imaging determination of Fe3+ and L-AA
Notes and references
We used SMMC-7721 cells as a model to study the Fe3+ and L-AA
dependent uorescence imaging of Y-CDs using a laser scan-
ning confocal microscope (LSCM). SMMC-7721 cells were
labelled with Y-CDs. As depicted in Fig. 7A, when Fe3+ entered Y-
CD-stained SMMC-7721 cells, the yellow uorescence was
gradually weakened and can hardly be seen aer 90 s.
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Sequentially, when L-AA was added the yellow emission was
progressively recovered in 180 s, indicating that the emission of
Y-CDs/Fe3+ in the cells can be recovered by L-AA. Fig. 7B shows
the morphology of cells cultured in the three cases of Y-CDs, Y-
CDs/Fe3+, and Y-CDs/Fe3+/L-AA. These cells emit orange (rst
panels in Fig. 7B), yellow (second panels in Fig. 7B), and blue
(third panels in Fig. 7B) uorescence when excited by 405, 488,
and 514 nm lasers, respectively. The cells cultured with Y-CDs
and Y-CDs/Fe3+/L-AA emit stronger uorescence than those
cultured with Y-CDs/Fe3+. The whole process shows that CDs
have an excellent ability to label cells. Fig. 7C depicts the
changes of the intracellular uorescence intensities of Y-CDs
without and with Fe3+ and L-AA, respectively. It is prominent
that the emission intensities of the cells are in the order of Y-
CDs>Y-CDs/Fe3+/L-AA>Y-CDs/Fe3+. The results illustrate that Y-
CDs can be used as nanoprobe to “on–off–on” detect Fe3+ and
L-AA in living cells.
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We develop concentration-dependent orange–yellow–blue uo-
rescent CDs for colorimetric and uorescent dual-mode detec-
tions of Fe3+ and L-AA. The concentration-dependent
uorescent CDs are prepared in a facile and eco-friendly
manner by hydrothermal treatment of coffee, salvia, and urea.
The as-prepared CDs exhibit low toxicity and excellent optical
stabilities. The uorescence intensity of Y-CDs is sensitive
toward Fe3+ and L-AA, which are utilized as a nanoprobe for “on–
off–on” determination of Fe3+ and L-AA. Meanwhile, a visible
color change is observed during the determination of Fe3+ and L-
AA. More importantly, Y-CDs could be successfully applied for
imaging intracellular Fe3+ and L-AA. By the combination of
uorescent and colorimetric readouts, Y-CDs could be poten-
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Conflicts of interest
There are no conicts to declare.
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Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (21575084) and the Natural Science Foundation
of Shanxi Province (201701D121019 and 201701D121017). We
also acknowledge Dr Ying Zuo for her help with AFM measure-
ments and Dr Juanjuan Wang for her help with Zeiss LSM 880
confocal laser-scanning microscope measurements from the
Scientic Instrument Center at Shanxi University.
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