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.