Sulfur and nitrogen binary doped carbon dots derived from ammonium thiocyanate for selective probing doxycycline in living cells and multicolor cell imaging

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Author’s Accepted Manuscript
Sulfur and nitrogen binary doped carbon dots
derived from ammonium thiocyanate for selective
probing doxycycline in living cells and multicolor
cell imaging
Mingyue Xue, Liangliang Zhang, Zhihua Zhan,
Mengbing Zou, Yong Huang, Shulin Zhao
To appear in:
Received date: 28 October 2015
Revised date:
9 December 2015
Accepted date: 10 December 2015
Cite this article as: Mingyue Xue, Liangliang Zhang, Zhihua Zhan, Mengbing
Zou, Yong Huang and Shulin Zhao, Sulfur and nitrogen binary doped carbon
dots derived from ammonium thiocyanate for selective probing doxycycline in
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Title: Sulfur and nitrogen binary doped carbon dots derived from 
ammonium thiocyanate for selective probing doxycycline in 
living cells and multicolor cell imaging 
Mingyue Xue,
Liangliang Zhang,*
Zhihua Zhan,
Mengbing Zou,

Yong Huang,
Shulin Zhao*

Key Laboratory for the Chemistry and Molecular Engineering of 
Medicinal Resources (Ministry of Education), College of Chemistry 
and Pharmacy, Guangxi Normal University, Guilin, 541004, China 
Guilin Normal College, Guilin, 541001, China. 
Corresponding author:
Professor Shulin Zhao, Liangliang Zhang
College of Chemistry and Pharmacy
Guangxi Normal University
Guilin, 541004, China 
Tel: +86 773 5845973 
Fax: +86 773 5832294 

A novel sulfur and nitrogen binary doped carbon dots (S,N-CDs) was synthetized by 
one-step manner through the hydrothermal treatment of citric acid (CA) and 
ammonium thiocyanate, and the procedures for biomedical applications, including 
probing doxycycline in living cells and multicolor cell imaging were developed. The 
obtained S,N-CDs are stable in aqueous solution, possess a very high quantum yield 
(QY, 74.15%) and good photostability. The fluorescence of S,N-CDs can be 
specifically quenched by doxycycline, providing a convenient turn-off assay of 
doxycycline. This assay shows a wide linear detection range from 0.08 to 60 μM with 
a low detection limit of 20 nM. The present method also displays a good selectivity. 
More importantly, the S,N-CDs have an excellent biocompatibility and low 
cytotoxicity, allowing the multicolor cell imaging and doxycycline detection in living 
cells. Consequently, the developed doxycycline methods is facile, low-cost, 
biocompatible, sensitive and selective, which may hold the potential applications in 
the fields of food safety and environmental monitoring, as well as cancer therapy and 
related mechanism research. 
Keywords: Sulfur; Nitrogen; Doped carbon dots; Multicolor cell imaging; 
Doxycycline detection

1. Introduction 
Doxycycline, a tetracycline derivative with a wide range of antibacterial activities, 
is frequently used to treat many infections such as chronic prostatitis, respiratory tract 
infections, sinusitis [1] and sexually transmitted diseases [2]. Additionally, it also 
applied in the fields of veterinary medicine and animal nutrition. But the abuse of 
doxycycline may lead to an unsafe residue level in the environment or food samples. 
To protect the safety of consumers, the European Union, America, Canada and China 
have legally set the maximum residue level for various antibiotics. Consequently, 
developing efficiently methods for the detection of doxycycline concentration is of 
great importance in pharmaceutical preparations, food safety and environmental 
monitoring. In the past 20 years, numerous analytical techniques including 
chromatography [3-5], capillary electrophoresis [6], spectrophotometry [7, 8], 
fluorescence [9], electrochemistry [10, 11], coupled technique [12-16] were used for 
doxycycline monitoring. However, these methods commonly suffered from low 
sensitivity and/or complicated preparation. In addition to the anti-microbial activity, 
doxycycline also shows an anti-tumour property in the treatment of prostate, 
pancreatic and colon cancer [17-19]. Thus, detecting doxycycline in cancer cells may 
provide more useful and direct information for cancer therapy and related mechanism 
research. But the reports about doxycycline monitoring in living cells are still rare at 
As fascinating carbon nanomaterials, fluorescent carbon dots (CDs) have attracted 
lots of research interests due to their excellent photostability, favorable 

biocompatibility, low toxicity without heavy metal ions and toxic elements, tunable 
fluorescence emission and excitation, and large Stokes shifts [18]. Based on these 
outstanding properties, CDs have been widely applied in the fields of bioimaging, 
biosensor and drug delivery [20-28]. Additionally, at present, great efforts were made 
to develop heteroatom-doped CDs for improving the optical and electrical properties 
of CDs. Boron, nitrogen, fluorine and sulfur were the commonly introduced doping 
chemical elements [29, 30]. Among these heteroatom-doped CDs, sulfur and nitrogen 
co-doped CDs were extremely attractive, which exhibited a high QY [31-33]. 
In this work, a novel low-cost strategy is developed for the synthesis of S,N-CDs 
by using CA and ammonium thiocyanate as precursors. The CA serves as the carbon 
source, while the ammonium thiocyanate provides sulfur and nitrogen. The 
as-obtained S,N-CDs are stable in aqueous solution and exhibit a very high QY 
(74.15%). The fluorescence can be specifically quenched by doxycycline. Thus, using 
S,N-CDs as fluorescent probes, a simple doxycycline detection was achieved (Scheme 
1). The high QY of obtained S,N-CDs offers a high sensitive for doxycycline assay. 
There are few works about the CDs-based detection of tetracyclines [34,35], but each 
tetracycline derivative could induce a similar response signal in these works. The 
proposed S,N-CDs probe here exhibits an approving selectivity and can distinguish 
between doxycycline and tetracycline. The high sensitivity and selectivity may ensure 
a more accurate analysis of doxycycline in complex samples. Remarkably, the 
synthesized S,N-CDs possess high photostability and excellent biocompatibility, good 
stability under the entire physiological pH range and high ionic strength. Coupled 

with these outstanding features, S,N-CDs are promising in the cell imaging and 
doxycycline monitoring in living cells. 
2. Experimental 
2.1. Reagents and materials
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased 
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Other chemical materials 
were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). All reagents 
were of analytical grade and were used without further purification. Water was 
purified with a Milli-Q plus 185 equip from Millipore (Bedford, MA, USA) and used 
throughout the work. 
2.2. Synthesis of S,N-CDs 
The S,N-CDs were prepared by thermal treatment of molecular organic salts with 
the mixed carbon source and the surface modifier in the single precursor. Briefly, 
2.1080 g CA and 1.5439 g of ammonium thiocyanate were dissolved into 5 mL water. 
The solutions were heated hydrothermally in a Teflon-equipped stainless-steel 
autoclave at 200 °C for 3 h with a heating rate of 10 °C min
. The obtained S, N-CDs 
solution was adjusted to pH 7 with 1 M NaOH solution and centrifuged at 10000 rpm 
for 20 min. A dialysis membrane (MWCO: 1 kDa; pore size: ca. 1.0 nm) was then 
used to separate the S,N-CDs from any residual unreacted species and obtained a 
brown solution. The solvent was removed with the aid of a rotary evaporator. Then 

the obtained S,N-CDs was dispersed into deionized (DI) water and preserved at a 4 °C 
for further characterization and use. 
2.3. Instruments and characterizations 
The morphology and microstructure of S,N-CDs were examined by high-resolution 
transmission electron microscopy (HRTEM) on a Tecnai G2 F20 microscope (FEI, 
Philips, Netherlands) with an accelerating voltage of 200 kV. The sample for HRTEM 
was made by dropping an aqueous solution onto a 300-mesh copper grid coated with a 
lacy carbon film. The X-ray diffraction (XRD) measurement was performed with a 
D/max-2500V/PC powder X-ray diffractometer (Rigaku,
Tokyo, Japan). The X-ray 
photoelectron spectroscopy (XPS) spectra of the sample were measured on a 
ESCALAB 250Xi X-ray Photoelectron Spectroscopy (Thermo Scientific, Waltham, 
MA, USA). Fourier transform infrared spectroscopy (FT-IR) study was conducted 
from KBr pellets on a PerkinElmer FT-IR spectrophotometer (Perkin-Elmer, Norwalk, 
Connecticut, USA). Elemental analyses (C, H, N, S) were carried out on a Perkin 
Elmer Series II CHNS/O 2400 elemental analyzer (Perkin-Elmer, Norwalk, 
Connecticut, USA). Fluorescence lifetime experiments were performed by a 
FL3-P-TCSPC time-resolved fluorescence spectrometer (Horiba Jobin Yvon,
Longjumeau, France). Raman spectra were obtained on a Raman Microscope 
(Renishaw, Gloucestershire, UK). All fluorescence spectra were obtained on a LS-55 
fluorescence spectrometer (Perkin-Elmer, Norwalk, Connecticut, USA). The emission 
spectra were recorded in the wavelength range of 400-600 nm. Ultraviolet–visible 
(UV-Vis) absorption spectra were characterized by a Cary 60 UV-Vis 

spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Cell imaging was 
examined by Zeiss LSM 710 confocal microscopy (Carl Zeiss, Oberkochen, 
2.4. Measurement of fluorescence QY 
Quinine sulfate (0.1 M H
as solvent; QY=0.54) was chosen as a standard. The 
QY of S,N-CDs (in water) were calculated according to the following equation: 



Where φ is the QY, Ι is the measured integrated emission intensity, η is the refractive 
index of the solvent, and A is the optical density. The subscript "st" refers to standard 
with known QY and "x" refers to the unknown samples. For these aqueous solutions, 

= 1. 
2.5. Detection of doxycycline 
Doxycycline samples with various concentration was incubated with 1 μg mL
S,N-CDs in 120 μL phosphate buffer (PB, 0.1 M, pH 6.0) at room temperature for 10 
min. The fluorescence measurements were then carried out. 
2.6. Cell imaging
Hepatocellular carcinoma cell (HepG2) were grown in a humidified atmosphere 
containing 5% CO
at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 
medium supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 μg mL
streptomycin and 100 units mL
penicillin. Cells were seeded on 35 mm glass bottom 
culture dishes for 24 h before confocal imaging. Then, the culture medium was 
replaced by 2.5 mL fresh medium containing 100 μg mL
S,N-CDs, and the cells 
were incubated for another 12 h at 37 °C under 5% CO
. The S,N-CDs labeled cells 

were then imaged by confocal microscopy. 
2.7. Cytotoxicity evaluation 
Cytotoxicity of S,N-CDs on cells was evaluated through MTT assay. HepG2 cells 
were seeded and incubated in a 96-well plate at a density of 1×10
cells per well for 
24 h. Then, the culture medium was removed, and the S,N-CDs were added into each 
well with the increasing concentrations from 100 to 500 μg mL
. The mixture was 
incubated for 24 h before replacing the medium with 200 μL fresh complete medium 
containing 20 μL MTT (5 mg mL
in PBS). After 4 h incubation, all medium was 
removed and 150 μL well
DMSO was added followed by shaking for 15 min. The 
absorbance of each well was measured at 570 nm using a using an Enzyme Linked 
Immunosorbent Assay reader with pure DMSO as a. blank. Non-treated cell was used 
as a control and the relative cell viability (mean% ± SD, n=3) was expressed as 
3. Results and discussion 
3.1. Synthesis and characterization of S,N-CDs 
The S,N-CDs were prepared by the hydrothermal treatment of CA and ammonium 
thiocyanate. Some synthetic conditions including the masses of CA and thiocyanate, 
the reaction time, and the reaction temperature were explored to obtain a higher QY. 
As shown in Table S1, the optimal conditions for S,N-CDs synthesis were set to be 
2.1080 g CA, 1.5439 g ammonium thiocyanate, and hydrothermal treatment at 200 °C 
for 3 h. The obtained S,N-CDs (QY 74.15%) under these conditions are used for the 

further characterization and application. 
The transmission electron microscopy (TEM) image (Fig. 1a) clearly indicates that 
the obtained S,N-CDs have a well dispersion and a diameter distribution between 4-9 
nm with an average size of ~7 nm. The high resolution TEM images display two 
lattice spacing of 0.23 and 0.32 nm, which may correspond to the (100) and (002) 
facet of graphite, respectively [31,36]. The disordered D band and crystalline G band 
are observed in the Raman spectra at 1348 cm
and 1573 cm
respectively (Fig. 1b). 
The intensity ratio between D band and G band is estimated to be 0.833, 
demonstrating that the S,N-CDs are highly crystalline and graphitic [37]. The X-Ray 
powder diffraction (XRD) pattern (Fig. 1C) shows a broader peak of (002) facet 
centered at about 25.5
, which is resulted from highly disordered carbon atoms and 
confirms the graphene structure of the obtained S,N-CDs [38]. 
The Fourier transform infrared (FTIR) spectra (Fig. S1) indicate the presence of 
N-H, S-H, oxygen-containing groups (O-H, -COO-, C=O, C-O), C-N and C-S in 
S,N-CDs. The doping of N and S atoms in the prepared S,N-CDs was also confirmed 
by XPS. The data in Fig. 2a clearly reveal the presence of C, O, N and S elements in 
the used CDs. The high resolution spectrum of C1s exhibits three main peaks (Fig. 
2b). The binding energy peak at 284.6 eV confirms the graphitic structure (sp2 C-C) 
of the S,N-CDs. The peak at about 285.4 eV suggests the presence of C-O, C-S, and 
C-N, and the peak around 288.4 eV could be assigned to C=O. The high resolution of 
the S2P XPS spectrum of S,N-CDs shows two peaks at 163.1, 164.3 (Fig. 2c), which 
are similar to the reported 2P
and 2P
positions of the–C–S– covalent bond of the 

thiophene-S. The high resolution N1s spectra (Fig. 2d) indicates the presence of both 
pyrrolic (400.1 eV) and graphitic (401.5 eV) N atoms [39]. Elemental analysis results 
further verify that the S,N-CDs are mainly composed of C (50.34%), O (35.27%, 
calculated), N (4.73%), S (6.68%), and H (2.98%). All of these demonstrate the 
successfully doping of N and S atoms into the framework of carbon dots. 
The UV-Vis spectrum shows two typical peaks centered at 232 and 335 nm, which 
are assigned to π→π* transition of C=C and n→π* transition of C=O, respectively. 
While the absorption of CA and ammonium thiocyanate is very weak at 232 or 335 
nm (Fig. 3, curves a and b). It is reported that the adsorption at 232 nm leads to nearly 
no observed fluorescence signal, and the adsorption at 335 nm due to the trapping of 
excited-state energy by the surface states results in strong emission [31]. 
Consequently, the light yellow S,N-CDs aqueous solution emitted a strong blue 
fluorescence under the irradiation at 365 nm (Fig. 3, inset). An excitation dependent 
emission wavelength and intensity was observed (Fig. S2). Furthermore, the 
fluorescence intensity was not significantly affected even after continuous irradiation 
at 365 nm UV light for 2 h or stored for 3 months (Fig. S3), while other conditions 
were kept unchanged. At the same time, the S,N-CDs solution exhibits a long-term 
homogeneous phase without any noticeable precipitation at room temperature. The 
fluorescence test of the S,N-CDs under different pH solutions indicates that the 
S,N-CDs are stable in the pH range from 3 to 11, and no obvious fluorescence change 
is observed (Fig. S4). The effect of ionic strength on the stability of S,N-CDs was also 
investigated by recording the fluorescence upon the addition of NaCl or KCl with 

different concentrations (from 0.25 M to 2.5 M) into the PB solution (pH 7.4). As 
shown in Fig. S5, the fluorescence intensities remained constant with the increase of 
ionic strength. As a result, according to the above discussion, the synthetized 
S,N-CDs have high QY, excellent photostability, good stability under the entire 
physiological pH range and high ionic strength, facilitating wider applications in 
sensing, labeling and imaging. 
3.2. Doxycycline detection 
The outstanding properties of S,N-CDs motivate us to explore their potential 
applications. A fluorescence quenching effect of doxycycline on S,N-CDs was 
observed. As shown in Fig. 4 (inset), the bright blue fluorescence of S,N-CDs is 
significantly quenched after the addition of doxycycline. Corresponding emission 
spectra also show that 80% of fluorescence intensity at 450 nm was lost in the 
presence of 60 μM doxycycline. In order to further understand the quenching 
mechanism, fluorescence lifetime and fluorescence intensity-based Stern-Volmer plot 
of S,N-CDs in the presence of doxycycline were investigated. As shown in Fig. S6, 
the plots of the F
/F versus doxycycline concentration is nearly fitted to the 
Stern-Volmer equation (F
[Q]+1), where F
and F are the fluorescence 
intensity of S,N-CDs in the absence and presence of doxycycline respectively, [Q] is 
the doxycycline concentration and K
is the Stern-Volmer constant. The average 
fluorescence lifetime for S,N-CDs is 2.89 ns, and it was about 2.86 ns in the presence 
of doxycycline. The addition of doxycycline did not significantly change the 
fluorescence lifetime (Fig. S7), indicating a static quenching process [36]. 

Before the doxycycline detection, several experiments were carried out to achieve 
the best assay performance. The effect of pH on the doxycycline assay was 
investigated by recording the fluorescence intensity under different pH values with 
and without doxycycline. A maximum fluorescence quenching was obtained when the 
pH value was adjusted to 6.0 (Fig. S8). Thus, a 0.1 M PB buffer with a pH value of 
6.0 was applied for this sensing system. The quenching time was also optimized. As 
shown in Fig.S9, about 80% fluorescence quenched was achieved when the 
doxycycline was added into the solution at 1 min, and the fluorescence kept stable 
during the following 10 min. Thus, to obtain effective quenching, 5 min was used as 
the optimal quenching time. This also indicated that the detection method is fast and 
easy by simply mixing the target and S,N-CDs. 
The sensitivity of the fabricated S,N-CDs fluorescent probe for doxycycline assay 
was then investigated by analyzing doxycycline with different concentrations under 
the optimal conditions. Fig. 5 shows the relationship between fluorescence quenching 
efficiency (F
and doxycycline concentrations. It can be found that the 
fluorescence quenching efficiency increased with the increase of doxycycline 
concentration. And in the doxycycline concentration range from 0.08 to 60 μM (0.037 
-28 μg mL
), the value of (F
was linear to the concentration of doxycycline 
(Fig. 5, inset). The linear regression was represented by the following equation: 
= 0.01312 C+0.00657 (R
=0.9985, C is the concentration of doxycycline). 
The detection limit (defined as 3σ, where σ is the standard deviation of the blank) was 
20 nM (~0.009 μg/mL). Compared to the most previous methods (Table S2), the 

proposed probes have wider linear detection range, and the detection limit is much 
The selectivity of the prepared S,N-CDs for doxycycline detection was investigated 
by testing the fluorescence response to some other antibiotics or biomolecules. Some 
antibiotics which were commonly found in food or environmental samples, including 
ampicillin, penicillin, streptomycin, tetracycline, were added in S,N-CDs-contained 
solutions. As shown in Fig. 6, under a large concentration (60 μM), only doxycycline 
rather than other antibiotics could induce a remarkable fluorescence quenching. 
Additionally, some other usually co-existed foreign biomolecules in biological or 
pharmaceutical samples, such as such as glucose, ascorbic acid, albumin human 
serum, glutathione and amino acids, were also tested. As show in Fig. S10, these 
foreign species have negligible effects on the fluorescence of S,N-CDs. The above 
results demonstrated an excellent selectivity for doxycycline detection. It should note 
that tetracycline, a common interference in fluorescence-quenching-based 
doxycycline detection methods [34,35], did not cause a distinct fluorescence 
quenching. This implies that the present S,N-CDs probes can distinguish between 
doxycycline and tetracycline, may offering a more accurate analysis of doxycycline in 
food or environmental samples. 
3.3. Cytotoxicity and Cell imaging 
To explore the possible application of the C-dots in bioimaging, the cytotoxicity of 
the as-prepared S,N-CDs was evaluated by MTT assay using HepG2 cells. As shown 
in Fig. S11, after the incubation with S,N-CDs for 24 h, the cell viability was still 

larger than 90% even the concentration of S,N-CDs up to 500 μg mL
. It is worthy to 
note that this concentration of S,N-CDs is much higher and the incubation time is 
much longer than those required for the potential bioimaging application. These 
results confirmed the low toxic and excellent biocompatibility of the prepared 
Because of it’s small size, good photostability and excellent biocompatibility, the 
prepared S,N-CD holds great potential for bioimaging application. We then used 
S,N-CDs for cell imaging and intracellular doxycycline monitoring. Fig. 7 shows 
confocal microscopy images of HepG2 cells incubated with S,N-CDs for 12 h at 
37 ℃. The S,N-CDs-stained cells exhibited blue, green and red colors upon the 
respective excitation at 405 nm, 488 nm and 514 nm. This result also indicates that 
the fluorescence can be collected with a broad range of excitation wavelength, and the 
prepared S,N-CDs can be used as excellent optical imaging probes for the further 
multicolor application. 
The S,N-CDs were then applied for detecting doxycycline in living cells based on 
the doxycycline-induced fluorescence quenching. Exogenous doxycycline was 
introduced into the S,N-CDs-pretreated HepG2 cells. One can find in Fig. 8 that upon 
supplementing cells with 100 μM of doxycycline in the growth medium for 20 min at 
37 ℃, microscope images showed no intracellular fluorescence. This result indicates 
that the proposed S,N-CDs could serve as an fluorescent probes for the detection of 
doxycycline in living cells. 

4. Conclusion
In summary, we introduced a one-step synthesis of S,N-CDs by the hydrothermal 
treatment of CA and ammonium thiocyanate, and applied the S,N-CDs for 
doxycycline detection in living cells and multicolor cell imaging. The fluorescence of 
S,N-CDs can be specifically quenched by doxycycline. The present fluorescent 
doxycycline assay has several important features. First, the prepared S,N-CDs exhibit 
a very high QY (74.15%), ensures a high sensitivity for quenching-based doxycycline 
assay. The detection limit in this work is much lower than that of most previous 
methods. Second, this method displays a satisfying selectivity and it can be used to 
distinguish between doxycycline and different antibiotics or biomolecules, which may 
provide a more accurate analysis of doxycycline in complex samples. Third, the 
proposed detection method is fast, low-cost and facile. Fourth, the S,N-CDs possess a 
good biocompatibility and low cytotoxicity, facilitating doxycycline detection in 
living cells. These advantages implied that the present S,N-CD-based doxycycline 
detection method have a wonderful prospect in the fields of food safety and 
environmental monitoring, as well as cancer therapy and related mechanism research. 
Furthermore, the outstanding properties of as-prepared S,N-CDs in preparation, 
stability, photostability and biocompatibility also make the S,N-CDs to be a 
promising material for the other wider applications. 
Financial support from the Natural Science Foundation of China (21405025, 

(2014GXNSFBA118047), Key Laboratory for the Chemistry and Molecular 
Engineering of Medicinal Resources of Education Ministry (CMEMR2013-A12), The 
Scientific Research Project of Guangxi Higher Learning (KY2015YB368) and 
Guangxi Normal University (2013ZD002) is gratefully acknowledged. 
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Figure Captions
Scheme 1. Scheme 1 Schematic illustration of S,N-CDs preparation and doxycycline 
Fig. 1. (a) TEM images and size distribution of S,N-CDs. (b) Raman spectroscopy of 
the N,S-CDs. (c) XRD pattern of the N,S-CDs. 
Fig. 2. (a) XPS spectra of the S,N-CDs. (b) High resolution C1S peak of the S,N-CDs. 
(c) High resolution S2p peak of the S,N-CDs. (d) High resolution N1s peak of the 
Fig. 3. Fluorescence spectra of the S,N-CDs and UV-Vis absorption spectra of the CA 
(a), ammonium thiocyanate (b) and S,N-CDs (c). Inset: Photograph of the S,N-CDs 
solution under irradiation of natural light (left) and UV light (right). 
Fig. 4. The fluorescence spectra of the S,N-CDs in the absence (a) or presence (b) of 
doxycycline. Inset: photographs of the S,N-CDs solution in the absence (right) and 
presence (left) of 60 μM doxycycline under irradiation of UV light. 
Fig. 5. The relationship between quenching efficiency and the concentration of 
doxycycline. Inset shows the linear response range. 

Fig. 6. Comparison of fluorescence intensities of S,N-CDs in the presence of 60 μM 
doxycycline, 60 μM ampicillin, 60 μM penicillin, 60 μM streptomycin, and 60 μM 
Fig. 7. Confocal microscopy images of HepG2 cells incubated with 100 μg mL
S,N-CDs for 12 h. (a) fluorescent image excited with a 405 nm laser, (b) fluorescent 
image excited with a 488 nm laser, (b) fluorescent image excited with a 514 nm laser, 
and (d) merged image of a, b and c. 
Fig. 8. Confocal microscopy images of HepG2 cells incubated with 100 μg mL
S,N-CDs (a) and after addition of 100 μM of doxycycline (b). 
Fig. 1. 

Fig. 2. 

Fig. 3. 

Fig. 4. 

Fig. 5. 
Fig. 6. 

Fig. 7. 

Fig. 8. 

Scheme 1 
Graphical Abstract

sulfur and nitrogen binary doped carbon dots derived from ammonium 
thiocyanate was synthesized.

► The sulfur and nitrogen binary doped carbon dots can be used for multicolor cell 
► High selective and sensitive method for doxycycline detection in living cells was 

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