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Carbon dots derived from rose flowers for tetracycline sensing
Talanta 140 (2015) 128–133
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Carbon dots derived from rose flowers for tetracycline sensing Yuanjiao Feng, Dan Zhong, Hong Miao, Xiaoming Yang n College of Pharmaceutical Sciences, Engineer Research Center of Chongqing Pharmaceutical Process and Quality Control, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 17 March 2015 Accepted 22 March 2015 Available online 27 March 2015
Herein, an innovative and simple method for synthesizing carbon dots (CDs) with satisfactory fluorescence has been successfully established while rose flowers served as carbon source for the first time. Meanwhile, the fluorescence (FL) mechanism of current CDs was elucidated in detail by fluorescence, UV–vis, HR-TEM, and FTIR-based analyses. Subsequently, this type of CDs was employed for detecting tetracycline (TC) on the basis of the interactions between TC and CDs, and allowed quenching their fluorescence. Moreover, the proposed analytical strategy permitted detecting TC in a linear range of 1.0 10 8 1.0 10 4 mol/L with a detection limit of 3.3 10 9 mol/L at a signal-to-noise ratio of 3. Significantly, the CDs described here were further applied for fluorescent coding, demonstrating their promising future towards various applications in analytic science. & 2015 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Rose flowers Tetracycline Detection
1. Introduction Carbon nanomaterials, mainly including carbon nanotubes, fullerenes, graphene, and carbon nanofilms, are promising scaffolds with fantastic sizes less than 10 nm [1], and have been playing critical roles in various fields such as biological markers [2], biochemistry and biomedicine [3]. Recently, appearances of fluorescent carbon dots (CDs) have exhibited tremendous impact on the advancement of various fields including electronics, photonics, energy, catalysis, and medicine [4], and attracted increasing interests owing to their less-toxicity, biocompatibility [5], photostability [5–9] and ease of preparation [10,11]. Thereby, CDs have been considered as a promising choice in potential applications for biosensing [12], catalysis [13], and imaging [14–16]. For example, CDs were utilized for conventional bioimaging of Ehrlich ascites carcinoma cells [17]. Meanwhile, a variety of methods for preparing CDs have been demonstrated in the past decades on account of their advanced characteristics, including electrochemical oxidation processes [18,19], arc discharge [20], hydrothermal cutting strategies [21], chemical oxidation methods [1,22,23], combustion/ thermal, and so on. Nevertheless, some of the proposed methods above required high costs, tedious steps, or special equipment, thus developing simple and rapid methods for synthesizing CDs are still meaningful. n
Corresponding author. Tel./fax: þ 86 23 68251225. E-mail address: [email protected] (X. Yang).
http://dx.doi.org/10.1016/j.talanta.2015.03.038 0039-9140/& 2015 Elsevier B.V. All rights reserved.
Tetracyclines (TCs), as effective antibiotics, have been widely used in the therapy of human and animal infections by Grampositive, Gram-negative bacteria and show effective activity for rickettsia, viral infection on the basis of their broad spectrum activity against pathogenic microorganisms, nice oral absorption, and relatively less toxicity and low cost [24,25]. However, irrational abusing of TCs may lead to increasing potential risk by residues in human body. To date, TCs abuse exist in our daily food such as milk [26] and honey [27]. Worse still, a large amount of data has demonstrated that long-term and repeated intake of TCs may affect the growth and formation of teeth. For the purpose of monitoring TCs, diverse techniques such as high performance liquid chromatography (HPLC) [28], chemiluminescence [29], capillary electrophoresis (CE) [30], and dipstick colorimetric methods [31] have been developed. Howbeit, most of these approaches exhibited more or less defects of sophisticated and costly instruments or complicated processes. Thus, the new and efficient methods for analyzing TCs remain necessary. Hereby, a simple and efficient method for synthesizing CDs has been built up by using rose flowers as carbon source for the first time (Fig. 1), which showed blue fluorescence together with a quantum yield of 13.45%. Subsequently, we applied this type of CDs for sensitively and selectively assaying TC based on the mechanism that the energies of CDs were captured for forming new bonds between the surface groups of TC and CDs [32]. In addition, the synthesized CDs were employed for detecting TC residues in human urine samples, suggesting their practicability. In short, we have broadened a new road for sensing and fluorescent staining.
Y. Feng et al. / Talanta 140 (2015) 128–133
129
2. Experimental
2.2. Instrumentation
2.1. Chemicals and materials
All fluorescence data were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were taken by a TECNAI G2 F20 microscope (Portland, America) at 200 kV and Fourier Transform Infrared Spectroscopy (FTIR) spectra were recorded by SHIMADZU IRprestige-21 spectrometer (Tokyo, Japan). Elemental and functional groups analyses were obtained by ESCALAB 250 X-ray photoelectron spectrometer. The quantum yields were obtained by using Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). The powder of CDs was obtained by lyophilisation in PiloFD8-4.3V (California, USA). The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Gongyi, China). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure the pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was used to blend the solution.
The rose flowers were bought from flower market (Chongqing, China). Phosphorus pentoxide (P2O5), TCs including tetracycline (TC), oxytetracycline (OTC), aureomycin, and doxycycline (DOXC), streptomycin, lincomycin, cysteine, histidine, glutathione, ibuprofen, ammonium thiocyanate, glucose, p-aminobenzoic acid, procaine were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Ascorbic acid, disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), sodium chloride (NaCl) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ, produced with an Aquapro AWL-0502-P ultrapure water system (Chongqing, China) was applied to all the following experiments.
2.3. Synthesis of CDs
Fig. 1. Schematic illustration of synthesizing CDs based on rose flowers.
The rose flowers as the carbon resource were first applied to synthesize CDs here. Briefly, rose flowers were on baking in the oven with 60° until the petals can be ground into powder. Then, 10 mL ultrapure water was added into the mixture of 10 mg petals
Fig. 2. (A) Fluorescence excitation and emission spectra and UV–vis spectrum of CDs. Inset: photographs of CDs solution (I,II); (B) emission spectra of CDs for varying excitation wavelengths; (C) HR-TEM images of CDs; (D) size distribution analysis of CDs. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Y. Feng et al. / Talanta 140 (2015) 128–133
Fig. 3. (A) The stability of CDs in different concentrations of NaCl ranging from 0.1 M to 1.0 M; (B) fluorescence variation of CDs for 100 min (record for every 5 min); (C) the stability of CDs in different pH solutions; (D) optimizing various scales of P2O5 (varying from 0.5 g to 4 g) for synthesizing CDs.
powder and 2.5 g P2O5, then stirring and standing for 10 min. Subsequently, the solution was put into the microwave oven for continuous heating till the color of solution changes from red to brown before exposure itself to room temperature and subjected it to concentration with 5000 rpm for 5 min. Finally, the CDs were filtered with 0.22 μm filter membrane to remove unnecessary products and further purified through a dialysis membrane (1000MWCO) for 24 h before use. 2.4. Detection of TC and interference experiments As a standard run, 40 μL CDs solution, 40 L phosphate buffer solution (10 mM, pH¼4) and various concentrations of TCs working solutions or sample solutions were added into a 1.5 mL vial successively. Subsequently, the reaction liquid was diluted to 400 μL with ultrapure water, and followed by vortex-mixing. The mixture was then incubated to react at 50 °C for 25 min, and subjected for fluorescence measurements. Towards the interference, stock solutions of similar substances (streptomycin, lincomycin, cysteine, GSH, vitamin C, histidine, ibuprofen, ammonium thiocyanate, glucose, p-aminobenzoic acid, procaine) were prepared. Additionally, interference originated from other chemicals were investigated individually in the presence of the CDs synthesized here, followed by the same process as TC detection. 2.5. Preparing urine samples Fresh human urine sample was collected from Southwest University Hospital for the recovery experiment. The urine samples were
subjected to centrifugation for 5 min, 5000 rpm. Finally, the supernatant was collected into 3 tubes and supplemented with standard TC solutions (0.8 10 4 M, 1.0 10 4 M, and 1.2 10 4 M).
3. Results and discussion 3.1. Characterization of CDs To investigate the synthesized CDs, the maximum excitation and emission spectra were initially recorded as 390 nm and 435 nm respectively, and the fluorescent properties of the CDs solution were subsequently described. As shown in Fig. 2A, the CDs aqueous solution showed obvious blue emission (photograph II) under UV light (365 nm) while appearing as yellowish and transparent under daylight (photograph I). To address whether the synthesis of CDs showed excitation-dependent emission character or not, different emission spectra upon varying excitation wavelengths were recorded. Briefly, the emission peak shifted to the longer wavelength and its corresponding fluorescence intensity decreased with the excitation wavelengths varying from 320 nm to 410 nm (Fig. 2B), and the CDs exhibited an obvious peak around 390 nm. Moreover, the morphology and particle size distributions of CDs were directly described by a high resolution transmission electron microscope (HR-TEM). As indicated in Fig. 2C and D, a majority population of CDs synthesized here existed within the size range of 4–6 nm and no aggregation emerged, depicting their satisfactory dispersity. For characterizing the surface groups and structure of the as-prepared CDs, FTIR was employed (Fig. S1).
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Specifically, O–H, C–H, C ¼ O, and C–O groups existed on the surface of CDs. Specifically, the absorption bands of O–H stretching vibrations emerged at 3420 cm 1 with C–H and stretching vibrations at 2930 cm 1. Similarly, the peaks at 1645 cm 1 and 1066 cm 1 were associated with C¼O and C–O stretching vibrations respectively. Towards the purpose of gaining insight into components of the current CDs, XPS survey spectra were further performed. As shown in Fig. S2, the three peaks at 522.7 eV, 400.9 eV and 290.8 eV were attributed to C 1s, N 1s, and O 1s respectively, suggesting that there mainly exist elements of C, N, and O for the carbon dots described here. Interestingly, benefited from their advantage of the prepared CDs, CDs were further employed for meaningful applications. As shown in Fig. 5D, four fluorescent paintings UV light stained with CDs were provided, implying their favorable fluorescent properties. Hence, these photographs suggested the possibility of these CDs for fluorescent staining.
3.3. Selectivity of the proposed method
3.2. Stability of CDs
3.4. Building up the assay for TC
Due to the importance of CDs' stability, corresponding experiments were conducted. As demonstrated in Fig. 3A–C, the fluorescent intensities of CDs hardly exhibited variations along with varying concentrations of NaCl, pH, and continuous excitation indicating their outstanding stability and tolerance of the CDs. In addition, various amounts of P2O5 (varying from 0.5 g to 4.5 g) were taken consideration for preparing CDs. As revealed in Fig. 3D, the fluorescent intensities of CDs exhibited variations along with different quantities of P2O5, suggesting that the property of CDs depended on the amount of P2O5 during the synthesis process, thus 2.5 g was selected as the optimal condition.
Towards the purpose of identifying the optimized conditions for detections, various factors possibly showing effect were explored. As the different pH may play a critical role to obtain the maximal decrease of the fluorescence intensity, various pH values were introduced during detecting TC. As shown in Fig. 5A, the fluorescence intensity decrease of CDs reached the maximum in the presence of TC as well as pH 4.0. Likewise, considering the possible influence of incubation time on the detecting procedures, varying time (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min) for reacting between CDs and TC has been
Considering their superior fluorescent properties of the CDs, we addressed whether they can be used as a fluorescent probe. As shown in Fig. 4A, the fluorescence intensity of the CDs at 435 nm decreased to about 50% once 100 mM TC was added. Simultaneously, the fluorescence decrease was obviously observed in the presence of 100 mM TC (picture b) compared with that in the absence of TC (picture a) under UV light, indicating the CDs showed the possibility for detecting TC. Likewise, the selectivity of this type of CDs was evaluated by testing the response to other reagents (500 mM for each) for the case of 100 mM tetracycline antibiotics (Fig. 4B). Obviously, tetracycline antibiotics rather than other antibiotics or chemicals showed much more responses to the fluorescent probe, indicating the favorable selectivity of CDs for assaying tetracycline antibiotics. As a result, TC was chosen as a representative for testing this fluorescent probe.
Fig. 4. (A) Fluorescence emission spectra of CDs in the absence (a) and presence (b) of TC (100 μM). Inset: Visual observation; (B) effect of different reagents on the fluorescence intensity of CDs.
Fig. 5. Optimizing the analysis conditions including pH (A), incubation time (B), and incubation temperature (C) in the absence (black) and presence (red) of TC (100 μM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. (A) Fluorescent spectra of CDs in the presence of various concentrations of TC; (B) Inset: plot of (F0–F) versus the logarithm of concentrations of TC introduced.
Table 1 Recoveries of TC supplemented in urine samples detected by the proposed method. Sample
TC supplemented (10 4 M)
TC measured (10 4 M)
Recovery (%)
1 2 3
1.2 1.0 0.8
1.18 0.978 0.81
98.3 97.8 101.25
the interactions between the surface groups of CD sand TC. And these interactions contributed to the energy of CDs captured by forming new bonds [32], thus facilitating to quench the fluorescence of CDs. Interestingly, the new type of CDs was employed for fluorescent staining, demonstrating their possibility to broaden avenues in biosensing and other purpose.
Acknowledgments also studied. In Fig. 5B, the fluorescence of CDs constantly decreased while the reaction time reached 25 min. Similarly, effects of reaction temperature ranging from 30 to 80 °C were studied in the presence of 100 mM TC separately (Fig. 5C), demonstrating that ΔF of CDs were also indeed dependent on reaction temperature. Eventually, pH 4.0, 50 °C, 25 min served as the optimal conditions during all the following experiment procedures. Furthermore, the calibration curve has been established upon the optimal conditions. Specifically, the fluorescent intensity decrease (F0–F) versus the logarithmic plot of TC concentrations displayed a linear range from 1.0 10 8 mol/L to 1.0 10 4 mol/L (Fig. 6). As depicted in Fig. 6B, (F0–F) was in the linear relationship along with concentrations of TC, and its related linear regression equation was (F0–F)¼75.0011xþ689.62CTC with a correlation coefficient of 0.9561 (n¼ 5), demonstrating the satisfactory precision of this fluorescent probe. Additionally, the detection limit of TC was 3.3 10 9 mol/L at a signal-to-noise ratio of 3. Taken together, these results suggested a promising method for assaying TC. 3.5. Detections of TC in real samples For investigating the practical application of the proposed method, standard recovery experiments were accordingly performed using human urine samples (Table 1). As listed, the recoveries of three samples were 98.3%, 97.8%, and 101.25%, indicating the current approach may broaden ways for practically detecting TC in real samples.
4. Conclusions In summary, we have creatively synthesized CDs originated from rose flowers according to a simple and green approach. Significantly, the CDs prepared here were applied as a biosensor for the quantification of TC on the basis of TC induced fluorescence quenching of CDs. This mechanism was presumably explained by
We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2015A005 and 2362014xk07), and Innovative Research Project for Postgraduate Student of Chongqing (CYS14049).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at: http://dx.doi.org/10.1016/j.talanta.2015.03. 038.
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Carbon dots derived from rose flowers for tetracycline sensing Yuanjiao Feng, Dan Zhong, Hong Miao, Xiaoming Yang n College of Pharmaceutical Sciences, Engineer Research Center of Chongqing Pharmaceutical Process and Quality Control, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 17 March 2015 Accepted 22 March 2015 Available online 27 March 2015
Herein, an innovative and simple method for synthesizing carbon dots (CDs) with satisfactory fluorescence has been successfully established while rose flowers served as carbon source for the first time. Meanwhile, the fluorescence (FL) mechanism of current CDs was elucidated in detail by fluorescence, UV–vis, HR-TEM, and FTIR-based analyses. Subsequently, this type of CDs was employed for detecting tetracycline (TC) on the basis of the interactions between TC and CDs, and allowed quenching their fluorescence. Moreover, the proposed analytical strategy permitted detecting TC in a linear range of 1.0 10 8 1.0 10 4 mol/L with a detection limit of 3.3 10 9 mol/L at a signal-to-noise ratio of 3. Significantly, the CDs described here were further applied for fluorescent coding, demonstrating their promising future towards various applications in analytic science. & 2015 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Rose flowers Tetracycline Detection
1. Introduction Carbon nanomaterials, mainly including carbon nanotubes, fullerenes, graphene, and carbon nanofilms, are promising scaffolds with fantastic sizes less than 10 nm [1], and have been playing critical roles in various fields such as biological markers [2], biochemistry and biomedicine [3]. Recently, appearances of fluorescent carbon dots (CDs) have exhibited tremendous impact on the advancement of various fields including electronics, photonics, energy, catalysis, and medicine [4], and attracted increasing interests owing to their less-toxicity, biocompatibility [5], photostability [5–9] and ease of preparation [10,11]. Thereby, CDs have been considered as a promising choice in potential applications for biosensing [12], catalysis [13], and imaging [14–16]. For example, CDs were utilized for conventional bioimaging of Ehrlich ascites carcinoma cells [17]. Meanwhile, a variety of methods for preparing CDs have been demonstrated in the past decades on account of their advanced characteristics, including electrochemical oxidation processes [18,19], arc discharge [20], hydrothermal cutting strategies [21], chemical oxidation methods [1,22,23], combustion/ thermal, and so on. Nevertheless, some of the proposed methods above required high costs, tedious steps, or special equipment, thus developing simple and rapid methods for synthesizing CDs are still meaningful. n
Corresponding author. Tel./fax: þ 86 23 68251225. E-mail address: [email protected] (X. Yang).
http://dx.doi.org/10.1016/j.talanta.2015.03.038 0039-9140/& 2015 Elsevier B.V. All rights reserved.
Tetracyclines (TCs), as effective antibiotics, have been widely used in the therapy of human and animal infections by Grampositive, Gram-negative bacteria and show effective activity for rickettsia, viral infection on the basis of their broad spectrum activity against pathogenic microorganisms, nice oral absorption, and relatively less toxicity and low cost [24,25]. However, irrational abusing of TCs may lead to increasing potential risk by residues in human body. To date, TCs abuse exist in our daily food such as milk [26] and honey [27]. Worse still, a large amount of data has demonstrated that long-term and repeated intake of TCs may affect the growth and formation of teeth. For the purpose of monitoring TCs, diverse techniques such as high performance liquid chromatography (HPLC) [28], chemiluminescence [29], capillary electrophoresis (CE) [30], and dipstick colorimetric methods [31] have been developed. Howbeit, most of these approaches exhibited more or less defects of sophisticated and costly instruments or complicated processes. Thus, the new and efficient methods for analyzing TCs remain necessary. Hereby, a simple and efficient method for synthesizing CDs has been built up by using rose flowers as carbon source for the first time (Fig. 1), which showed blue fluorescence together with a quantum yield of 13.45%. Subsequently, we applied this type of CDs for sensitively and selectively assaying TC based on the mechanism that the energies of CDs were captured for forming new bonds between the surface groups of TC and CDs [32]. In addition, the synthesized CDs were employed for detecting TC residues in human urine samples, suggesting their practicability. In short, we have broadened a new road for sensing and fluorescent staining.
Y. Feng et al. / Talanta 140 (2015) 128–133
129
2. Experimental
2.2. Instrumentation
2.1. Chemicals and materials
All fluorescence data were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cell. Meanwhile, UV/vis spectra were recorded by a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were taken by a TECNAI G2 F20 microscope (Portland, America) at 200 kV and Fourier Transform Infrared Spectroscopy (FTIR) spectra were recorded by SHIMADZU IRprestige-21 spectrometer (Tokyo, Japan). Elemental and functional groups analyses were obtained by ESCALAB 250 X-ray photoelectron spectrometer. The quantum yields were obtained by using Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). The powder of CDs was obtained by lyophilisation in PiloFD8-4.3V (California, USA). The thermostatic water bath (DF-101s) was purchased from Gongyi Instrument Co., Ltd. (Gongyi, China). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure the pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was used to blend the solution.
The rose flowers were bought from flower market (Chongqing, China). Phosphorus pentoxide (P2O5), TCs including tetracycline (TC), oxytetracycline (OTC), aureomycin, and doxycycline (DOXC), streptomycin, lincomycin, cysteine, histidine, glutathione, ibuprofen, ammonium thiocyanate, glucose, p-aminobenzoic acid, procaine were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Ascorbic acid, disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), sodium chloride (NaCl) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MΩ, produced with an Aquapro AWL-0502-P ultrapure water system (Chongqing, China) was applied to all the following experiments.
2.3. Synthesis of CDs
Fig. 1. Schematic illustration of synthesizing CDs based on rose flowers.
The rose flowers as the carbon resource were first applied to synthesize CDs here. Briefly, rose flowers were on baking in the oven with 60° until the petals can be ground into powder. Then, 10 mL ultrapure water was added into the mixture of 10 mg petals
Fig. 2. (A) Fluorescence excitation and emission spectra and UV–vis spectrum of CDs. Inset: photographs of CDs solution (I,II); (B) emission spectra of CDs for varying excitation wavelengths; (C) HR-TEM images of CDs; (D) size distribution analysis of CDs. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Y. Feng et al. / Talanta 140 (2015) 128–133
Fig. 3. (A) The stability of CDs in different concentrations of NaCl ranging from 0.1 M to 1.0 M; (B) fluorescence variation of CDs for 100 min (record for every 5 min); (C) the stability of CDs in different pH solutions; (D) optimizing various scales of P2O5 (varying from 0.5 g to 4 g) for synthesizing CDs.
powder and 2.5 g P2O5, then stirring and standing for 10 min. Subsequently, the solution was put into the microwave oven for continuous heating till the color of solution changes from red to brown before exposure itself to room temperature and subjected it to concentration with 5000 rpm for 5 min. Finally, the CDs were filtered with 0.22 μm filter membrane to remove unnecessary products and further purified through a dialysis membrane (1000MWCO) for 24 h before use. 2.4. Detection of TC and interference experiments As a standard run, 40 μL CDs solution, 40 L phosphate buffer solution (10 mM, pH¼4) and various concentrations of TCs working solutions or sample solutions were added into a 1.5 mL vial successively. Subsequently, the reaction liquid was diluted to 400 μL with ultrapure water, and followed by vortex-mixing. The mixture was then incubated to react at 50 °C for 25 min, and subjected for fluorescence measurements. Towards the interference, stock solutions of similar substances (streptomycin, lincomycin, cysteine, GSH, vitamin C, histidine, ibuprofen, ammonium thiocyanate, glucose, p-aminobenzoic acid, procaine) were prepared. Additionally, interference originated from other chemicals were investigated individually in the presence of the CDs synthesized here, followed by the same process as TC detection. 2.5. Preparing urine samples Fresh human urine sample was collected from Southwest University Hospital for the recovery experiment. The urine samples were
subjected to centrifugation for 5 min, 5000 rpm. Finally, the supernatant was collected into 3 tubes and supplemented with standard TC solutions (0.8 10 4 M, 1.0 10 4 M, and 1.2 10 4 M).
3. Results and discussion 3.1. Characterization of CDs To investigate the synthesized CDs, the maximum excitation and emission spectra were initially recorded as 390 nm and 435 nm respectively, and the fluorescent properties of the CDs solution were subsequently described. As shown in Fig. 2A, the CDs aqueous solution showed obvious blue emission (photograph II) under UV light (365 nm) while appearing as yellowish and transparent under daylight (photograph I). To address whether the synthesis of CDs showed excitation-dependent emission character or not, different emission spectra upon varying excitation wavelengths were recorded. Briefly, the emission peak shifted to the longer wavelength and its corresponding fluorescence intensity decreased with the excitation wavelengths varying from 320 nm to 410 nm (Fig. 2B), and the CDs exhibited an obvious peak around 390 nm. Moreover, the morphology and particle size distributions of CDs were directly described by a high resolution transmission electron microscope (HR-TEM). As indicated in Fig. 2C and D, a majority population of CDs synthesized here existed within the size range of 4–6 nm and no aggregation emerged, depicting their satisfactory dispersity. For characterizing the surface groups and structure of the as-prepared CDs, FTIR was employed (Fig. S1).
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Specifically, O–H, C–H, C ¼ O, and C–O groups existed on the surface of CDs. Specifically, the absorption bands of O–H stretching vibrations emerged at 3420 cm 1 with C–H and stretching vibrations at 2930 cm 1. Similarly, the peaks at 1645 cm 1 and 1066 cm 1 were associated with C¼O and C–O stretching vibrations respectively. Towards the purpose of gaining insight into components of the current CDs, XPS survey spectra were further performed. As shown in Fig. S2, the three peaks at 522.7 eV, 400.9 eV and 290.8 eV were attributed to C 1s, N 1s, and O 1s respectively, suggesting that there mainly exist elements of C, N, and O for the carbon dots described here. Interestingly, benefited from their advantage of the prepared CDs, CDs were further employed for meaningful applications. As shown in Fig. 5D, four fluorescent paintings UV light stained with CDs were provided, implying their favorable fluorescent properties. Hence, these photographs suggested the possibility of these CDs for fluorescent staining.
3.3. Selectivity of the proposed method
3.2. Stability of CDs
3.4. Building up the assay for TC
Due to the importance of CDs' stability, corresponding experiments were conducted. As demonstrated in Fig. 3A–C, the fluorescent intensities of CDs hardly exhibited variations along with varying concentrations of NaCl, pH, and continuous excitation indicating their outstanding stability and tolerance of the CDs. In addition, various amounts of P2O5 (varying from 0.5 g to 4.5 g) were taken consideration for preparing CDs. As revealed in Fig. 3D, the fluorescent intensities of CDs exhibited variations along with different quantities of P2O5, suggesting that the property of CDs depended on the amount of P2O5 during the synthesis process, thus 2.5 g was selected as the optimal condition.
Towards the purpose of identifying the optimized conditions for detections, various factors possibly showing effect were explored. As the different pH may play a critical role to obtain the maximal decrease of the fluorescence intensity, various pH values were introduced during detecting TC. As shown in Fig. 5A, the fluorescence intensity decrease of CDs reached the maximum in the presence of TC as well as pH 4.0. Likewise, considering the possible influence of incubation time on the detecting procedures, varying time (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min) for reacting between CDs and TC has been
Considering their superior fluorescent properties of the CDs, we addressed whether they can be used as a fluorescent probe. As shown in Fig. 4A, the fluorescence intensity of the CDs at 435 nm decreased to about 50% once 100 mM TC was added. Simultaneously, the fluorescence decrease was obviously observed in the presence of 100 mM TC (picture b) compared with that in the absence of TC (picture a) under UV light, indicating the CDs showed the possibility for detecting TC. Likewise, the selectivity of this type of CDs was evaluated by testing the response to other reagents (500 mM for each) for the case of 100 mM tetracycline antibiotics (Fig. 4B). Obviously, tetracycline antibiotics rather than other antibiotics or chemicals showed much more responses to the fluorescent probe, indicating the favorable selectivity of CDs for assaying tetracycline antibiotics. As a result, TC was chosen as a representative for testing this fluorescent probe.
Fig. 4. (A) Fluorescence emission spectra of CDs in the absence (a) and presence (b) of TC (100 μM). Inset: Visual observation; (B) effect of different reagents on the fluorescence intensity of CDs.
Fig. 5. Optimizing the analysis conditions including pH (A), incubation time (B), and incubation temperature (C) in the absence (black) and presence (red) of TC (100 μM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. (A) Fluorescent spectra of CDs in the presence of various concentrations of TC; (B) Inset: plot of (F0–F) versus the logarithm of concentrations of TC introduced.
Table 1 Recoveries of TC supplemented in urine samples detected by the proposed method. Sample
TC supplemented (10 4 M)
TC measured (10 4 M)
Recovery (%)
1 2 3
1.2 1.0 0.8
1.18 0.978 0.81
98.3 97.8 101.25
the interactions between the surface groups of CD sand TC. And these interactions contributed to the energy of CDs captured by forming new bonds [32], thus facilitating to quench the fluorescence of CDs. Interestingly, the new type of CDs was employed for fluorescent staining, demonstrating their possibility to broaden avenues in biosensing and other purpose.
Acknowledgments also studied. In Fig. 5B, the fluorescence of CDs constantly decreased while the reaction time reached 25 min. Similarly, effects of reaction temperature ranging from 30 to 80 °C were studied in the presence of 100 mM TC separately (Fig. 5C), demonstrating that ΔF of CDs were also indeed dependent on reaction temperature. Eventually, pH 4.0, 50 °C, 25 min served as the optimal conditions during all the following experiment procedures. Furthermore, the calibration curve has been established upon the optimal conditions. Specifically, the fluorescent intensity decrease (F0–F) versus the logarithmic plot of TC concentrations displayed a linear range from 1.0 10 8 mol/L to 1.0 10 4 mol/L (Fig. 6). As depicted in Fig. 6B, (F0–F) was in the linear relationship along with concentrations of TC, and its related linear regression equation was (F0–F)¼75.0011xþ689.62CTC with a correlation coefficient of 0.9561 (n¼ 5), demonstrating the satisfactory precision of this fluorescent probe. Additionally, the detection limit of TC was 3.3 10 9 mol/L at a signal-to-noise ratio of 3. Taken together, these results suggested a promising method for assaying TC. 3.5. Detections of TC in real samples For investigating the practical application of the proposed method, standard recovery experiments were accordingly performed using human urine samples (Table 1). As listed, the recoveries of three samples were 98.3%, 97.8%, and 101.25%, indicating the current approach may broaden ways for practically detecting TC in real samples.
4. Conclusions In summary, we have creatively synthesized CDs originated from rose flowers according to a simple and green approach. Significantly, the CDs prepared here were applied as a biosensor for the quantification of TC on the basis of TC induced fluorescence quenching of CDs. This mechanism was presumably explained by
We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2015A005 and 2362014xk07), and Innovative Research Project for Postgraduate Student of Chongqing (CYS14049).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at: http://dx.doi.org/10.1016/j.talanta.2015.03. 038.
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