Contents
1. Introduction Page No.2
2. Experimental Section Page No.3
2.1. Chemicals and materials Page
No.3
2.2. Instrumentation Page No.3
2.3. Synthesis of CDs Page No.3
2.4. CDs for pH sensing Page No.3
2.5. Optimization of CDs detecting tigecycline Page No.3
2.6. Detection of tigecycline and the lake-water sample Page No.3
3. Results and discussion Page No.3
3.1. Preparation of CDs Page No.3
3.2. Characterization of CDs Page No.4
3.3. Fluorescence stability of CDs Page No.4
3.4. Exploration of CDs sensing pH and the corresponding mechanism Page
No.4
3.5. Establishing the assay strategy of tigecycline by CDs Page No.5
3.6. Optimizing the conditions of CDs detecting tigecycline
Page No.5
3.7. Anti-interference and selectivity for CDs detecting tigecycline
Page No.5
3.8. Detection of tigecycline in the lake water sample Page No.5
3.9. Detection mechanism of tigecycline by CDs Page No.5
4. Conclusions Page No.6
Acknowledgement Page No.6
References Page No.6
1. Introduction
Carbon dots (CDs) exhibit their own superiority among kinds of
fluorescent materials due to the low excellent luminescence, synthetic
cost, satisfactory water solubility and biosafety [1,
2]. Thus, they have been widely utilized in various fields of
fluorescence sensing [3,4], bio-imaging[5], drug delivery [6] and
even more. Howbeit, most CDs only produce the fluorescence emissions
with the short wavelengths, which may be difficult to avoid the effect
of biological autofluorescence [7-9], thus
restricting their application. Therefore, the preparation of CDs with
long wavelength emission is essential and challengeable[10,11]. As been acknowledged, doping N atoms is
an effective approach to achieve their fluorescent red-shift of CDs
owing to the similar atomic size of C and N atoms[12]. In addition, other doping elements such as S[13] and Se [14] also play a
critical role towards the emissions red-shifting of CDs.
As been well-known, pH plays an important role during numerous
detections and biological processes such as the apoptosis and ion
transport [15,16], thus various methods have been
developed for pH detection [17,18]. CDs are also
considered as one of the ideal candidates for pH sensing due to their
abundant functional groups, which usually show the obvious response to
the variation of pH. Consequently, developing the innovative CDs to
detecting pH values is still meaningful.
The family of tetracycline antibiotics have been mainly utilized for
treating animal diseases by virtue of their broad-spectrum antibacterial
activity and low cost [19]. However, their over
residuals in the environment could induce the direct ecosystem
contamination. In addition, their excessive accumulation in food such as
pork and milk also show the damage to human health, leading to the
endocrine disorders, central nervous system defects and beyond[20]. Tigecycline (TGC), being a member of
tetracycline antibiotics, still serves during the therapy towards the
critical diseases owing to its low antibiotic resistance[21]. Nevertheless, the abusing of tigecycline
could also lead to the carcinogenesis and malformation, even increase
the mortality risk of the patients [22].
Therefore, it is valuable of developing the ways to detect tigecycline.
Herein, we originally synthesized CDs with the bright yellow
fluorescence by using alizarin and ethylenediamine through the
hydrothermal method. To be specific, the CDs well dispersed and
exhibited the advanced long-wavelength emission. Importantly, the
introduction of tigecycline obviously quenched their fluorescence of
CDs, thus providing a new strategy of detecting tigecycline, and also we
explored and elucidated the mechanism of CDs sensing tigecycline.
Meanwhile, we employed the CDs prepared here for pH sensing in the
acidic range based on the varied fluorescence of CDs accompanied by pH
decreasing.
2. Experimental Section
2.1. Chemicals and materials
Typically, alizarin, ethylenediamine, tigecycline, tetracycline,
oxytetracycline, chlortetracycline, doxycycline, minocycline, isoniazid,
clindamycin, gentamicin, chloramphenicol, D-penicillamine and urea were
purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China)
as well as uric acid, L-cysteine, L-arginine, L-histidine, glucose from
Aladdin Reagent Co., Ltd. (Shanghai, China). Similarly, peroxide,
hydrochloric acid, sodium hydroxide, magnesium sulfate, acetic acid,
methanol, ethanol, isopropyl alcohol, tetrahydrofuran and N,
N-dimethylacetamide were acquired from Chron Chemicals Co., Ltd.
(Chengdu, China). Again, the ultrapure water of 18.25 MΩ cm was provided
by an Aquapro AWL-0502-P ultrapure water system (Chongqing, China),
while Britton-Robinson (BR) buffer served for regulating the pH values
of various solutions.
2.2. Instrumentation
Initially, the fluorescent measurements were performed with a Hitachi
fluorescence spectrophotometer of F-7000 (Tokyo, Japan), while the
UV-vis absorption spectra were recorded using a Shimadzu
spectrophotometer of UV-1750 (Tokyo, Japan). Subsequently, the
investigation of elements and functional groups were achieved by X-ray
photoelectron spectrometer of ESCALAB 250 (New York, USA) and a
Prestige-21 Fourier transform infrared (FTIR) spectrometer (Tokyo,
Japan). Further, the transmission electron microscopy (TEM) images were
obtained with a TECANI G2 F20 microscope (Columbia, America), and we
introduce Zetasizer Nano Malvern laser particle sizer (Malvern, England)
to identify the particle sizes. The vortex mixer of QL-901 and
thermostatic air drying oven of DHG-9140A (Shanghai, China) were
employed during the process of preparation CDs, and we used the acidity
meter pHS-3C for obtaining different pH. Again, the centrifuge (TG16-W)
of Cence instrument Co. Ltd. (Changsha, China) was utilized to separate
the mixtures. Then, the related powder of CDs was collected by
SCIENTZ-12N (Ningbo, China) through lyophilization.
2.3. Synthesis of CDs
In this work, the CDs were synthesized through the hydrothermal reaction
of alizarin and ethylenediamine in ultrapure water. The optimal
conditions for synthesizing CDs were determined by regulating the
reaction temperature and time. Specifically, 0.09 g of alizarin was
dissolved in 10 mL of ultrapure water, and 50 μL of ethylenediamine was
added to the solution with vortex mixing. Then, this mixture was
transferred to a 25 mL of high-pressure reactor and reacted at 200 °C
for 6 h. After cooling to room temperature, the brown solution was
collected and subjected to the centrifugation for removing the
precipitation. And the obtained supernatant was diluted with 20 mL of
ultrapure water, then the undissolved particles were removed through the
filtration with a 0.22 μM of water-soluble microporous filter membrane.
Further, the filtered solution was purified by using the dialysis bag of
MWCO:1000 Da for 24 h, and the final product of the yellow powder was
acquired through lyophilization.
2.4. CDs for pH sensing
Basically, the CDs were dissolved in 10 mL of ultrapure water to obtain
a concentration of 15 μg/mL, and the pH range from 2 to 12 of these
paralleled solutions were adjusted by adding hydrochloric acid and
sodium hydroxide.
2.5. Optimization of CDs detecting tigecycline
To obtain the optimal assay conditions for the detection of tigecycline,
the potential effect of the incubation temperature and time were
explored. Particularly, 15.0 mg of CDs powder was dissolved in 100 mL of
PBS buffer (pH=7.4), and the tigecycline solution of 0.02 mol/L was also
prepared with PBS buffer during the process of optimizing the detection
conditions. Next, 100 μL of CDs, 100 μL of tigecycline and 800 μL of PBS
buffer were sequentially added to the paralleled EP tubes, and the
mixtures were placed in a water bath at different temperatures of 20 °C,
25 °C, 30 °C, 35 °C, 40 °C, 45
°C, 50 °C and 60 °C for 15 minutes followed by their fluorescence
intensity recorded immediately. Similarly, these mixtures were incubated
at the optimal detection temperature for 10, 20, 30, 40, 50, 60, 70 and
80 minutes, and their fluorescence was instantly detected. Accordingly,
the fluorescence variation between the mixtures and the CDs only with
the same conditions was calculated, and the conditions with the largest
difference were identified as the optimal detection conditions.
2.6. Detection of tigecycline and the lake-water sample
At the beginning, 15.0 mg of CDs powder was dissolved in 100 mL of PBS
buffer (pH=7.4), and each CDs solution of 100 μL was separately mixed
with 800 μL of PBS buffer (pH=7.4) and 100 μL of tigecycline with
different concentrations. Subsequently, the corresponding fluorescence
intensity of each mixture was measured at the excitation of 470 nm after
the incubation at room temperature for 10 minutes. Towards verifying the
practicality of the assay, the obtained lake-water sample was
centrifuged at 10,000 rpm for 20 minutes, and the supernatant was
collected and stored at 4 °C. Then the supernatant was supplemented by
tigecycline with different concentrations, and the related fluorescence
was detected followed by calculating the recovery of tigecycline.
3. Results and discussion
3.1. Preparation of CDs
Basically, the CDs described here were synthesized by the hydrothermal
method, and the optimal preparation conditions were acquired by
regulating the reaction temperature and time. In brief, the fluorescence
intensity of CDs increased with the reaction temperature and time
increasing, and CDs showed the highest fluorescence with the reaction
time of 6 h at 200 °C (Figure S1). Thus, 200 °C and 6 h were determined
as the optimized conditions.
3.2. Characterization of CDs
Initially, the fluorescence performance of CDs prepared here was
investigated, and their emission was 546 nm as well as the excitation
wavelength of 470 nm (Figure 1A and 1B). Meanwhile, the solution of CDs
exhibited the bright yellow fluorescence with UV of 365 nm (Figure 1A,
inset b) and the light yellow under daylight (Figure 2A, inset a).
Again, the emission wavelengths of CDs varied with various excitation
wavelengths, revealing their fluorescent excitation-dependent property
(Figure 1C).
To deeply understand the CDs prepared here, a series of systematic
characterizations was performed to explore their morphology and
structure. As shown in Figure 1D, the CDs uniformly dispersed and showed
spherical with an average particle size of 4.64 nm, and the crystal
lattice of CDs was 0.23 nm, which was consistent with the typical
morphological characteristic of CDs. Simultaneously, the dynamic light
scattering (DLS) data showed that the particle size distribution of CDs
ranged from 2.68 nm to 7.1 nm (Figure 1E), which mainly agreed with the
result obtained by HR-TEM. Besides, the UV absorption spectrum of CDs
showed two peaks at 261 nm and 472 nm, attributing to the π-π*
transition of aromatic C=C as well as the n-π* transition of C=O (Figure
1F). Meanwhile, the XRD pattern of CDs showed a peak at 22.6° (Figure
1G), corresponding to the (111) facet of graphite and revealing the
existence of the highly disordered carbon atoms[23,24].
Further, Fourier transform infrared (FTIR) and X-ray photoelectron
spectroscopy (XPS) were employed to identify the functional groups and
elemental composition of CDs. As shown in Figure 1H, the broad
absorption band in the range of 2750 to 3750 cm-1indicated the existed groups of O-H or N-H (3445 cm-1and 3223 cm-1) for CDs [25,26].
The absorption peak near at 2400 cm-1 was mainly
contributed to S-H stretching vibration and the peaks at 1622
cm-1 and 1471 cm-1 were originated
from the stretching vibrations of C=O and C-N, respectively[27,28]. Meanwhile, the peak at 1037
cm-1 was generated by the stretching vibration of C-O[29-31], and the peak located at 653
cm-1was ascribed to C-S. Thus, the FTIR spectrum of
CDs proved that they were doped with the elements of N and S.
Furthermore, the XPS spectra revealed that the CDs we prepared were
composed of four major elements including C, N, O and S (Figure 1I).
Specifically, the spectrum of C 1s was divided as three peaks, which
were originated from C-C/C=C (284.77 eV), C-O/C-N (286.28 eV) and C=O
(288.92 eV), respectively (Figure S2A). Likewise, the N 1s spectrum was
fitted as three peaks for pyridinic N at 398.62 eV, amino N at 400.19 eV
and C-N at 401.57 eV, where pyridinic N could significantly reduce the
band gap, facilitating the CDs to show a longer emission wavelength
(Figure S2B). Again, the spectrum of S 2p exhibited two major peaks at
168.19 eV and 168.54 eV for SO32-(Figure S2C). Moreover, two peaks of O 1s were responsible for C=O
(531.59 eV) and C-O (533.09 eV), respectively (Figure S2D). Overall, the
FTIR and XPS characterizations of CDs demonstrated the existed groups of
carboxyl, hydroxyl, amino and sulfite on their surfaces.