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Multifunctional sensing applications of biocompatible N-doped carbon dots as pH and Fe3+ sensors
Microchemical Journal 149 (2019) 103981
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Multifunctional sensing applications of biocompatible N-doped carbon dots as pH and Fe3+ sensors
T
Xu Chen1, Jianliang Bai1, Yusha Ma, Guojun Yuan, Jing Mei, Lu Zhang, Lili Ren
⁎
School of Chemistry & Chemical Engineering, Southeast University, Nanjing 211189, China
ARTICLE INFO
ABSTRACT
Keywords: N-doped Long-wavelength emission Multifunctional sensors Cell imaging
Recently, carbon dots (CDs) with long wavelength (i.e., orange and red-light) are gradually critical in the bioimaging and biomedical fields. In this paper, a simple one-pot hydrothermal method for preparing bright orange N-doped carbon dots (N-CDs) from p-phenylenediamine has been developed. The resulting N-CDs with abundant surface functional groups show strong orange fluorescence, certain water solubility property and obvious fluorescence quenching to Fe3+ ions and pH. What's more, the fluorescence of the N-CDs displays a consequent color variation with the increasing concentration of Fe3+ and pH. In short, N-CDs can be considered as an efficient and multifunctional fluorescent sensor for monitoring pH and Fe3+ because of their prominent biocompatibility and low toxicity characteristics.
1. Introduction Recently, photoluminescent carbon dots (CDs) acted as an original kind of emerging nanocarbon have aroused considerable interest due to the excellent photoluminescence [1–4], abundant precursor sources [5], easy surface functionalization for diverse applications [6,7], good bio-compatibility and low toxicity [8–12]. All these unique advantages of CDs distinguish them from organic dyes and make them play significant role in many important fields, like fluorescence sensing, drug delivery, optoelectronic devices and bioimaging [10,13–18]. In previous reports, a majority of CDs usually emitted intense blue or green fluorescence, which influenced their performances in cellular imageforming due to their fragile penetration property and even serious optical destruction to biological cells [14,19–22]. Hence, the preparation of CDs with efficient long-wavelength fluorescence (especially red and orange) is apparently preferred to extend the practical applications of CDs. In recent years, some strategies have been made toward CDs with long-wavelength emission. For example, Jiang et al. prepared red, green and blue CDs by hydrothermal method and then proved their excellent multicolor fluorescence performance in biological imaging [14]. Xiong and colleagues prepared the N-Doped red CDs by using column chromatography method following the hydrothermal and successfully applied their CDs for cellular imaging, both in vitro and in vivo [20]. Zhan and colleagues synthesized multicolor CDs using 1,3,6-trinitropyrene
(TNP) as the precursors in different solutions in one pot and used for bioimaging applications in vitro and vivo [23]. Chen et al. synthesized the orange and blue dual-color CDs by using formamide as the reaction medium and applied to LEDs [21]. However, their CDs possessed poor soluble in water, which restricted their utilizations in bioimaging and biosensing [24,25]. Therefore, it is of a desperate need to synthesize red or orange emissive CDs with good water solubility. As is known to all, pH is an important value both in our surroundings and in biological processes [26–28]. Even small changes of pH could result in evident responses to our environment, animals and even ourselves. The pH measurement methods include NMR [29], electrochemistry [30], absorption [31] and fluorometric measurement [32–34] etc. Among different approaches to detect pH, using CDs with sufficient biosecurity is meaningful. Meanwhile, iron (Fe3+) is one of the important ions of biological body because Fe3+ exerts significant effect in biological fields by combining with all kinds of proteins [35–38]. Recently, a series of CDs have been synthesized for detecting Fe3+. Qu et al. prepared CNPs using dopamine as an effective fluorescent sensor for detecting Fe3+ sensitively [39]. Zhou and colleague prepared CDs in one pot based on the citric acid and tris precursors, which showed excellent selectivity for Fe3+ [40]. Jia et al. put forward an eco-friendly way to obtain N-CDs from black soya beans and exhibited ideal sensitivity of detection of Fe3+ [41]. However, despite many reports about CDs for pH sensing or Fe3+ detection, there are still few publications about double-mode nanoprobe for multi-functional
Corresponding author. E-mail address: [email protected] (L. Ren). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.microc.2019.103981 Received 24 March 2019; Received in revised form 4 June 2019; Accepted 4 June 2019 Available online 08 June 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Microchemical Journal 149 (2019) 103981
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then introduced into a 50 mL Teflon-lined stainless steel autoclave and heated at 180 °C for 8 h. After that, the mixture including N-CDs was purified with silica column chromatography utilizing ethanol as the eluent. Purified N-CDs of orange fluorescence were finally obtained which were dried under vacuum. 2.4. Fluorescence “turn-off” assay for sensing Fe3+ For Fe3+ measurement, the typical operation is as follows: 200 μL of N-CDs (1.0 mg/mL) were introduced into PBS buffer solutions (10 mM, pH = 7.1–7.4). Then, Fe3+ ions with various concentrations were mixed with the above solution to survey the detection range of Fe3+. Afterward, the fluorescence intensity was recorded Meanwhile, the other metal cations, for example, K+, Na+, Zn2+, Ni2+, Fe2+, Cd2+, Mn2+, Mg2+, Ba2+ and Ca2+ were added in to the above N-CDs solution, which were also recorded. During the measurement process, all the parameters kept same.
Scheme 1. Illustration of the synthetic procedure of the N-CDs and the N-CDs based fluorescence sensing platform for the detection of Fe3+ and pH.
2.5. Fluorescence assay of pH Briefly, pH value of N-CDs solution is mediated using 0.1 M phosphate buffer solution (PBS) (pH = 3.0–9.0) and 1 mol/L of HCl (pH = 1.0–2.0), respectively. Then, the fluorescence intensity of these solutions was measured.
sensing. Herein, we design to prepare a kind of N-CDs with certain water solubility through simple solvent thermal reaction, which have long-wavelength emission and can be used for both pH sensing and Fe3+ detection. And this will have important biological implications (Scheme. 1).
2.6. Cell imaging
2. Experimental
HeLa cells were dispersed in 24-well plates including 10% FBS and 5% CO2 at 37 °C. Then, the medium was substituted with N-CDs solution (1.0 mg/mL) and cultivated for 1.5 h. Afterwards, the cells were washed carefully using 2 mL PBS to clear vestigial N-CDs away. Cellular images were carried out with a confocal microscope. To evaluate the intracellular Fe3+ detection ability of N-CDs, the cells treated with NCDs and 200 μM Fe3+ were imaged.
2.1. Materials All the reagents were of analytical grade and used as received without further purification. P-Phenylenediamine and ammonia water (25%–28%) were obtained from Across Organics. NaOH and HCl used to adjust the pH were obtained from Beijing Chemical Works (Beijing, China). FeCl3, NaCl, KCl, BaCl2, CaCl2, MnCl2, CdCl2, ZnCl2, NiCl2, FeCl2 and MgCl2 were obtained from Aladdin. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from MCE. Fetal bovine serum (FBS) was purchased from BOVOGEN. Trypsin EDTA and Dulbecco's modified Eagle (DMEM) were purchased from GIBCO. Deionized water (DI) with the resistance of 18.1 MΩ was used throughout the experiments.
3. Results and discussion 3.1. Prepare and characterization of the N-doped C-dots The strong orange-fluorescence N-CDs were facilely prepared by one-pot method which only used p-phenylenediamines as carbon source and ammonia as nitrogen source. Ammonia was introduced as a kind of N dopant, because it is cheap and full of N atoms. Achieving this method is more attractive than other N-CDs preparation methods [25]. What's more, under the circumstance of hydrothermal condition, carbonization process happened to obtain N-CDs with enriched amino and hydroxyl groups. As the Fig. 1(a) displayed, the N-CDs are well dispersed and average size is nearly 1.5 nm. The HRTEM image demonstrates that N-CDs possess a lattice spacing of 0.21 nm (Fig. 1(b)), which is consistent with the (100) lattice distance of sp2 graphitic carbon as previous mentioned [42]. As the Raman image (Fig. S1) depicts, two obvious bands appear at approximately 1387 and 1567 cm−1, which should be assigned to N-CDs' the D and G bands, separately. Furthermore, the Raman shift of the N-CDs demonstrates the existence of NeH (1424 cm−1), C]C or C]N (1624 cm−1) of the phenazine structure [43]. In general, it is confirmed that N-CDs have some degree of graphitization. FT-IR and XPS spectra could further investigate chemical compositions of the N-CDs. The broad peak in the region of 3426–3305 cm−1 resulting from the stretching vibration of eNH and eOH indicates the existence of eNH and eOH groups in the FT-IR spectrum (Fig. S2). The absorption bands at around 1635 cm−1 and 1520 cm−1 should correspond to C]O and C]N bonds of benzene rings. The 1373 cm−1 peak may belong to the bending vibration of eNH (Fig. S2). The XPS survey spectrum indicates that the N-CDs primarily include carbon (74.93%), oxygen (19.5%) and nitrogen (5.56%) components (Fig. 2a). From the
2.2. Material characterization The structure and morphological features of N-CDs were investigated by Transmission Electron Microscopy (TEM) operating on a JEM-2011 microscope. Fourier transform infrared (FTIR) spectra were obtained by using a Bruker Tensor 27 ATR-FTIR spectrometer with KBr pellet. Ultraviolet–visible absorption spectra were obtained by using a Shimadzu UV-2600 spectrometer. Fluorescence emission and excitation spectra of N-CDs were measured by a Horiba Fluoromax-4 fluorescence spectrophotometer. The X-ray photoelectron spectra (XPS) were carried out on a Thermo ESCALAB 250XI electron spectrometer. Raman spectra were recorded on a Renishaw Raman system model 1000 spectrometer with λex = 532 nm. Fluorescence lifetimes were determined by a Fluorolog-3 spectrofluorometer. pH values were adjusted via using a PHS-3C pH meter (Leici, Shanghai). The cellular imaging was tested by confocal laser fluorescence microscopy (TCS SP5II, Leica, Germany). 2.3. Synthesis of carbon dots The N-CDs were prepared through a handy and one-step hydrothermal way. Typically, p-phenylenediamines (0.05 g) and ammonia water (100 μL) were transferred into 50 mL flask containing 10 mL ethanol. The mixture was stirred for about 1 h at room temperature, 2
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Fig. 1. TEM images and lateral size distribution diagrams of N-CDs.
C1s XPS spectrum (Fig. 2b), we can see that there are four peaks at 284.3, 284.7, 285.7 and 288.8 eV, corresponding to C]C, CeC, CeO/ CeN, C]O/C]N, respectively [44–46]. Simultaneously, the Fig. 2b also proves that nitrogen atom has been doped into N-CDs successfully. The corresponding deconvoluted N1 s spectrum exhibits three peaks of pyridinic type (398.2 eV), amino type (398.9 eV) and pyrrolic type (400.0 eV) N atoms which demonstrate that the surface of N-CDs is rich in -NH2 groups (Fig. 2c) [43]. The O1s XPS spectrum (Fig. 2d) exhibits two signals at 533.5 and 532.1 eV, which proved the existence of C]O and CeOH/CeOeC, respectively [20]. Thus, the above results indicate that N-CDs possess numerous multiple functional groups like eOH, eNH2, which consists with the results of FT-IR.
The UV–Vis absorption and PL spectra are depicted in Fig. 3. The two intense peaks (Fig. 3a) at 210 and 260 nm may arise from the π-π* transition of the C]C and the n-π* transition of the CeN bond and the contribution of surface sections. In the lower-energy region, the weak absorption bands from 400 nm to 450 nm may imply that N-CDs possess surface states. The N-CDs could emit visible bright orange fluorescence under UV light (Fig. 3a inset). The quantum yield of N-CDs is 15.8% utilizing Rhodamin 6G as a reference. In addition, a detailed survey about PL properties has been measured as the excitation wavelength is from 400 to 560 nm by an increment of 20 nm. The N-CDs show the typical excitation dependence characteristic (Fig. 3b), confirming that the surface state of the N-CDs may influence the band gap of N-CDs.
Fig. 2. The XPS spectra of the N-CDs and the high-resolution XPS C 1s, N 1s, and O 1s spectra of the selected samples. Each band was deconvoluted following the literature. 3
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Fig. 3. (a) UV–Vis absorption of N-CDs. The inset displays photographs of the N-CDs under daylight (left) and UV irradiation (right) inaqueous solution. (b) PL spectra of N-CDs at different λex400–560 nm.
3.2. Fluorescence response of N-doped C-dots toward pH
In addition, it is necessary to measure the pH-reversibility of the NCDs. Using 1 M HCl and 1 M NaOH to vary the pH value of the N-CDs aqueous solution back and forth, then the PL intensity change was inspected between acidic (pH = 2.0) and neutral (pH = 7.0) environment. It can be seen that (Fig. 4b) the PL intensity can successfully recover when the pH value is adjusted multiple times from 2.0 to 7.0. It is fairly visible that the N-CDs show good pH-reversibility.
pH value plays important role whether in our surroundings or biological organs, tissues and cells. It was imperative to investigate the possibility of the nitrogen doped CDs for pH sensing as a fluorescence probe. Thus, corresponding experiments were carried out for further evaluation the pH-dependent response of the N-CDs. As shown in Fig. S3 (a), the color variations of the N-CDs with the different pH solutions is from orange to brown and then to pale pink with the naked eye as the pH values change from 9.0 to 1.0. Meanwhile, in Fig. S3 (b), the color of solutions varies from bright orange to green and then to nearly colorless with the decreases of the pH from 9.0 to 1.0. Moreover, the pH -sensitive feature of N-CDs was examined (Fig. 4a). The PL intensity of the N-CDs exhibited hardly obvious variation which was maximum at pH 7.0 when the pH ranges from 9.0 to 4.0. But it is evident that the PL intensity decreases sharply at lower pH (1.0–3.0) value. It may be due to the fact that amine groups are more likely to be protonated under strong acid conditions which possibly induces the aggregation of N-CDs, leading to the fluorescence quenching [47–50]. At the same time, the generation of new surface state (NH3+) induces slight blue shift of the PL peaks when the pH is decreasing [51]. To sum up, the N-CDs could be served as the dual-mode pH signal sensing from both fluorescence intensity and color change. It is sensible and convenient to exploit this property to detect pH by identifying the color of N-CDs solution. What's more, the fluorescent luminescence characteristic of N-CDs solution is easily affected by the extreme acidic (pH 1.0–3.0) conditions corresponding to the pH value of human gastric juice. It is expected to be applied to detect stomach diseases.
3.3. Sensitive and selective detection of Fe3+ It is also very important to detect Fe3+ in water solutions since Fe3+ ion exists in large quantities in the environment and exerts vital role in many physiological and pathological fields by combining with various regulatory proteins. In this research, the sensitivity of N-CDs for detecting Fe3+ was investigated. Fig. S4 displays two photographs of NCDs solutions (1 mg·mL−1) of different Fe3+ concentrations under visible and UV light illumination. The fluorescence color of N-CDs (Fig. S4.b) is gradually changing from bright orange to yellow to grass green. Additionally, the color of the N- CDs solutions under sunlight (Fig. S4.a) is from light orange to brown. Moreover, in Fig. 5a, the PL intensity of N-CDs gradually decreases with the increasing Fe3+ concentrations (0–300 μM), supported by gradual color variation of N-CDs optical photos under 365 nm light excitation. Furthermore, the fluorescent quenching tendency exhibited a fine linear relationship (R2 = 0.9912) as the concentration of Fe3+ is in the range of 0–200 μM. The selectivity performance of the N-CDs is furthermore explored. It is evident that the N-CDs exhibit sensitive selectivity toward Fe3+ ions over other metal ions like K+, Na+, Zn2+, Ni2+, Mn2+, Mg2+, Fe2+,
Fig. 4. (a) Fluorescence spectra of the N-CDs dispersed different pH values (1.0–9.0); (b) the reversible PL intensity of N-CDs dispersion when the pH value alternates between 2.0 and 7.0. 4
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Fig. 5. (a) Fluorescent emission spectra of the N-CDs in the presence of different concentrations of Fe3+from 0 to 300 μM. (b) The fluorescent responses to various metal ions.
Fig. 6. Sensing principle of nanoprobes for Fe3+.
Fig. 7. (a) Fluorescence decay of N-CDs in the absence of 200 μM Fe3+; (b) Fluorescence decay of N-CDs in the presence of 200 μM Fe3+.
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Cd2+, Ca2+, Ba2+ and Fe3+ metal ions. As exhibited in Fig. 5b, it's clearly illustrated that the other interference metal ions exhibited no evident effect on the fluorescence intensities of the N-CDs. However, once Fe3+ ion is added in the above testing solutions, the PL intensities of the N-CDs is significantly quenched. From the above results, it was proposed a possible mechanism to explain quenching mechanism of NCDs by Fe3+ ion (Fig. 6). The fluorescent quenching effect of N-CDs may result from the complexes of N-CDs and Fe3+ ion. It is known that the Fe3+ ion with half-filled 3d orbital could form the effective coordination with the various functional groups (e.g., hydroxyl, amino) on the surface of N-CD through strong interaction [52]. Especially, N atom on the surface of the N-CDs can serve as electron donors, which is also critical for increasing the local electronic density of the N-CDs. Such interactions facilitate the abundant electrons in the excited state of N-CDs transferring to the 3d orbital of Fe3+ and finally cause the strong fluorescence quenching. To further elucidate the detection mechanism, the PL lifetime is measured. As shown in Fig. 7, the PL lifetime of the N-CDs reduces from 7.14 ns to 6.09 ns when 200 μM Fe3+ ion was added. It suggests that the fluorescence quenching is dynamic, which clearly illustrates an ultrafast excitation recombination process.
fluorescence under 405 nm excitation wavelength which clearly indicates that the N-CDs own outstanding cell permeability. In the second investigation, HeLa cells incubating with the N-CDs/Fe3+ (200 μM) for 1.5 h (Fig. 8(d–f)) emits grass green fluorescence. These phenomena demonstrate that the N-CDs prepared could be applied for the cellular imaging with low cytotoxicity and certain water solubility. 4. Conclusion Overall, bright orange N-CDs were synthesized using the pPhenylendiamine as the precursor and ammonia as the nitrogen source by hydrothermal way. The as-prepared N-CDs exhibit excellent quantum yield (QY) (15.8%), abundant functional groups, outstanding biocompatibility and cell imaging ability. Such N-CDs can act as fluorescent nanosensors with multiple applications for detecting pH and Fe3+ ion selectively and sensitively. More importantly, the detection of pH values and Fe3+ was immediate by both visible and fluorescence variation. Furthermore, the N-CDs can be successfully introduced into HeLa cells to monitor intracellular Fe3+ on the basis of fluorescence intensity variation.
3.4. Biological application of N-CDs It is well known that excess and scarcity of Fe3+ could influence the cellular homeostasis, leading to various diseases. Thus, it's essential to further evaluate the feasibility of the N-CDs for imaging in living cells by the laser confocal microscope. The Fig. 8(a–c) represents the living image of HeLa cells cultivating with N-CDs (1.0 mg/ mL) for 1.5 h under corresponding bright-field, fluorescence and merged. As shown in the Fig. 8(a–c), the confocal microscopic images show bright orange
Acknowledgments We sincerely express gratitude to the Fundamental Research Funds for the Central Universities of China (2242016K41018 and KYLX16_0193) and A Project subsidized the Priority Academic Program Development of Jiangsu Higher Education Institutions (1107047002).
Fig. 8. Fluorescence microscopyimages for HeLa cells. (a–c) HeLacells incubated with the probe N-CDs (50 μg mL−1) for 1.5 h. (d–f) The cells were incubated with NCDs/Fe3+ (200 μM) for1.0 h.
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Appendix A. Supplementary data
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Multifunctional sensing applications of biocompatible N-doped carbon dots as pH and Fe3+ sensors
T
Xu Chen1, Jianliang Bai1, Yusha Ma, Guojun Yuan, Jing Mei, Lu Zhang, Lili Ren
⁎
School of Chemistry & Chemical Engineering, Southeast University, Nanjing 211189, China
ARTICLE INFO
ABSTRACT
Keywords: N-doped Long-wavelength emission Multifunctional sensors Cell imaging
Recently, carbon dots (CDs) with long wavelength (i.e., orange and red-light) are gradually critical in the bioimaging and biomedical fields. In this paper, a simple one-pot hydrothermal method for preparing bright orange N-doped carbon dots (N-CDs) from p-phenylenediamine has been developed. The resulting N-CDs with abundant surface functional groups show strong orange fluorescence, certain water solubility property and obvious fluorescence quenching to Fe3+ ions and pH. What's more, the fluorescence of the N-CDs displays a consequent color variation with the increasing concentration of Fe3+ and pH. In short, N-CDs can be considered as an efficient and multifunctional fluorescent sensor for monitoring pH and Fe3+ because of their prominent biocompatibility and low toxicity characteristics.
1. Introduction Recently, photoluminescent carbon dots (CDs) acted as an original kind of emerging nanocarbon have aroused considerable interest due to the excellent photoluminescence [1–4], abundant precursor sources [5], easy surface functionalization for diverse applications [6,7], good bio-compatibility and low toxicity [8–12]. All these unique advantages of CDs distinguish them from organic dyes and make them play significant role in many important fields, like fluorescence sensing, drug delivery, optoelectronic devices and bioimaging [10,13–18]. In previous reports, a majority of CDs usually emitted intense blue or green fluorescence, which influenced their performances in cellular imageforming due to their fragile penetration property and even serious optical destruction to biological cells [14,19–22]. Hence, the preparation of CDs with efficient long-wavelength fluorescence (especially red and orange) is apparently preferred to extend the practical applications of CDs. In recent years, some strategies have been made toward CDs with long-wavelength emission. For example, Jiang et al. prepared red, green and blue CDs by hydrothermal method and then proved their excellent multicolor fluorescence performance in biological imaging [14]. Xiong and colleagues prepared the N-Doped red CDs by using column chromatography method following the hydrothermal and successfully applied their CDs for cellular imaging, both in vitro and in vivo [20]. Zhan and colleagues synthesized multicolor CDs using 1,3,6-trinitropyrene
(TNP) as the precursors in different solutions in one pot and used for bioimaging applications in vitro and vivo [23]. Chen et al. synthesized the orange and blue dual-color CDs by using formamide as the reaction medium and applied to LEDs [21]. However, their CDs possessed poor soluble in water, which restricted their utilizations in bioimaging and biosensing [24,25]. Therefore, it is of a desperate need to synthesize red or orange emissive CDs with good water solubility. As is known to all, pH is an important value both in our surroundings and in biological processes [26–28]. Even small changes of pH could result in evident responses to our environment, animals and even ourselves. The pH measurement methods include NMR [29], electrochemistry [30], absorption [31] and fluorometric measurement [32–34] etc. Among different approaches to detect pH, using CDs with sufficient biosecurity is meaningful. Meanwhile, iron (Fe3+) is one of the important ions of biological body because Fe3+ exerts significant effect in biological fields by combining with all kinds of proteins [35–38]. Recently, a series of CDs have been synthesized for detecting Fe3+. Qu et al. prepared CNPs using dopamine as an effective fluorescent sensor for detecting Fe3+ sensitively [39]. Zhou and colleague prepared CDs in one pot based on the citric acid and tris precursors, which showed excellent selectivity for Fe3+ [40]. Jia et al. put forward an eco-friendly way to obtain N-CDs from black soya beans and exhibited ideal sensitivity of detection of Fe3+ [41]. However, despite many reports about CDs for pH sensing or Fe3+ detection, there are still few publications about double-mode nanoprobe for multi-functional
Corresponding author. E-mail address: [email protected] (L. Ren). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.microc.2019.103981 Received 24 March 2019; Received in revised form 4 June 2019; Accepted 4 June 2019 Available online 08 June 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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then introduced into a 50 mL Teflon-lined stainless steel autoclave and heated at 180 °C for 8 h. After that, the mixture including N-CDs was purified with silica column chromatography utilizing ethanol as the eluent. Purified N-CDs of orange fluorescence were finally obtained which were dried under vacuum. 2.4. Fluorescence “turn-off” assay for sensing Fe3+ For Fe3+ measurement, the typical operation is as follows: 200 μL of N-CDs (1.0 mg/mL) were introduced into PBS buffer solutions (10 mM, pH = 7.1–7.4). Then, Fe3+ ions with various concentrations were mixed with the above solution to survey the detection range of Fe3+. Afterward, the fluorescence intensity was recorded Meanwhile, the other metal cations, for example, K+, Na+, Zn2+, Ni2+, Fe2+, Cd2+, Mn2+, Mg2+, Ba2+ and Ca2+ were added in to the above N-CDs solution, which were also recorded. During the measurement process, all the parameters kept same.
Scheme 1. Illustration of the synthetic procedure of the N-CDs and the N-CDs based fluorescence sensing platform for the detection of Fe3+ and pH.
2.5. Fluorescence assay of pH Briefly, pH value of N-CDs solution is mediated using 0.1 M phosphate buffer solution (PBS) (pH = 3.0–9.0) and 1 mol/L of HCl (pH = 1.0–2.0), respectively. Then, the fluorescence intensity of these solutions was measured.
sensing. Herein, we design to prepare a kind of N-CDs with certain water solubility through simple solvent thermal reaction, which have long-wavelength emission and can be used for both pH sensing and Fe3+ detection. And this will have important biological implications (Scheme. 1).
2.6. Cell imaging
2. Experimental
HeLa cells were dispersed in 24-well plates including 10% FBS and 5% CO2 at 37 °C. Then, the medium was substituted with N-CDs solution (1.0 mg/mL) and cultivated for 1.5 h. Afterwards, the cells were washed carefully using 2 mL PBS to clear vestigial N-CDs away. Cellular images were carried out with a confocal microscope. To evaluate the intracellular Fe3+ detection ability of N-CDs, the cells treated with NCDs and 200 μM Fe3+ were imaged.
2.1. Materials All the reagents were of analytical grade and used as received without further purification. P-Phenylenediamine and ammonia water (25%–28%) were obtained from Across Organics. NaOH and HCl used to adjust the pH were obtained from Beijing Chemical Works (Beijing, China). FeCl3, NaCl, KCl, BaCl2, CaCl2, MnCl2, CdCl2, ZnCl2, NiCl2, FeCl2 and MgCl2 were obtained from Aladdin. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from MCE. Fetal bovine serum (FBS) was purchased from BOVOGEN. Trypsin EDTA and Dulbecco's modified Eagle (DMEM) were purchased from GIBCO. Deionized water (DI) with the resistance of 18.1 MΩ was used throughout the experiments.
3. Results and discussion 3.1. Prepare and characterization of the N-doped C-dots The strong orange-fluorescence N-CDs were facilely prepared by one-pot method which only used p-phenylenediamines as carbon source and ammonia as nitrogen source. Ammonia was introduced as a kind of N dopant, because it is cheap and full of N atoms. Achieving this method is more attractive than other N-CDs preparation methods [25]. What's more, under the circumstance of hydrothermal condition, carbonization process happened to obtain N-CDs with enriched amino and hydroxyl groups. As the Fig. 1(a) displayed, the N-CDs are well dispersed and average size is nearly 1.5 nm. The HRTEM image demonstrates that N-CDs possess a lattice spacing of 0.21 nm (Fig. 1(b)), which is consistent with the (100) lattice distance of sp2 graphitic carbon as previous mentioned [42]. As the Raman image (Fig. S1) depicts, two obvious bands appear at approximately 1387 and 1567 cm−1, which should be assigned to N-CDs' the D and G bands, separately. Furthermore, the Raman shift of the N-CDs demonstrates the existence of NeH (1424 cm−1), C]C or C]N (1624 cm−1) of the phenazine structure [43]. In general, it is confirmed that N-CDs have some degree of graphitization. FT-IR and XPS spectra could further investigate chemical compositions of the N-CDs. The broad peak in the region of 3426–3305 cm−1 resulting from the stretching vibration of eNH and eOH indicates the existence of eNH and eOH groups in the FT-IR spectrum (Fig. S2). The absorption bands at around 1635 cm−1 and 1520 cm−1 should correspond to C]O and C]N bonds of benzene rings. The 1373 cm−1 peak may belong to the bending vibration of eNH (Fig. S2). The XPS survey spectrum indicates that the N-CDs primarily include carbon (74.93%), oxygen (19.5%) and nitrogen (5.56%) components (Fig. 2a). From the
2.2. Material characterization The structure and morphological features of N-CDs were investigated by Transmission Electron Microscopy (TEM) operating on a JEM-2011 microscope. Fourier transform infrared (FTIR) spectra were obtained by using a Bruker Tensor 27 ATR-FTIR spectrometer with KBr pellet. Ultraviolet–visible absorption spectra were obtained by using a Shimadzu UV-2600 spectrometer. Fluorescence emission and excitation spectra of N-CDs were measured by a Horiba Fluoromax-4 fluorescence spectrophotometer. The X-ray photoelectron spectra (XPS) were carried out on a Thermo ESCALAB 250XI electron spectrometer. Raman spectra were recorded on a Renishaw Raman system model 1000 spectrometer with λex = 532 nm. Fluorescence lifetimes were determined by a Fluorolog-3 spectrofluorometer. pH values were adjusted via using a PHS-3C pH meter (Leici, Shanghai). The cellular imaging was tested by confocal laser fluorescence microscopy (TCS SP5II, Leica, Germany). 2.3. Synthesis of carbon dots The N-CDs were prepared through a handy and one-step hydrothermal way. Typically, p-phenylenediamines (0.05 g) and ammonia water (100 μL) were transferred into 50 mL flask containing 10 mL ethanol. The mixture was stirred for about 1 h at room temperature, 2
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Fig. 1. TEM images and lateral size distribution diagrams of N-CDs.
C1s XPS spectrum (Fig. 2b), we can see that there are four peaks at 284.3, 284.7, 285.7 and 288.8 eV, corresponding to C]C, CeC, CeO/ CeN, C]O/C]N, respectively [44–46]. Simultaneously, the Fig. 2b also proves that nitrogen atom has been doped into N-CDs successfully. The corresponding deconvoluted N1 s spectrum exhibits three peaks of pyridinic type (398.2 eV), amino type (398.9 eV) and pyrrolic type (400.0 eV) N atoms which demonstrate that the surface of N-CDs is rich in -NH2 groups (Fig. 2c) [43]. The O1s XPS spectrum (Fig. 2d) exhibits two signals at 533.5 and 532.1 eV, which proved the existence of C]O and CeOH/CeOeC, respectively [20]. Thus, the above results indicate that N-CDs possess numerous multiple functional groups like eOH, eNH2, which consists with the results of FT-IR.
The UV–Vis absorption and PL spectra are depicted in Fig. 3. The two intense peaks (Fig. 3a) at 210 and 260 nm may arise from the π-π* transition of the C]C and the n-π* transition of the CeN bond and the contribution of surface sections. In the lower-energy region, the weak absorption bands from 400 nm to 450 nm may imply that N-CDs possess surface states. The N-CDs could emit visible bright orange fluorescence under UV light (Fig. 3a inset). The quantum yield of N-CDs is 15.8% utilizing Rhodamin 6G as a reference. In addition, a detailed survey about PL properties has been measured as the excitation wavelength is from 400 to 560 nm by an increment of 20 nm. The N-CDs show the typical excitation dependence characteristic (Fig. 3b), confirming that the surface state of the N-CDs may influence the band gap of N-CDs.
Fig. 2. The XPS spectra of the N-CDs and the high-resolution XPS C 1s, N 1s, and O 1s spectra of the selected samples. Each band was deconvoluted following the literature. 3
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Fig. 3. (a) UV–Vis absorption of N-CDs. The inset displays photographs of the N-CDs under daylight (left) and UV irradiation (right) inaqueous solution. (b) PL spectra of N-CDs at different λex400–560 nm.
3.2. Fluorescence response of N-doped C-dots toward pH
In addition, it is necessary to measure the pH-reversibility of the NCDs. Using 1 M HCl and 1 M NaOH to vary the pH value of the N-CDs aqueous solution back and forth, then the PL intensity change was inspected between acidic (pH = 2.0) and neutral (pH = 7.0) environment. It can be seen that (Fig. 4b) the PL intensity can successfully recover when the pH value is adjusted multiple times from 2.0 to 7.0. It is fairly visible that the N-CDs show good pH-reversibility.
pH value plays important role whether in our surroundings or biological organs, tissues and cells. It was imperative to investigate the possibility of the nitrogen doped CDs for pH sensing as a fluorescence probe. Thus, corresponding experiments were carried out for further evaluation the pH-dependent response of the N-CDs. As shown in Fig. S3 (a), the color variations of the N-CDs with the different pH solutions is from orange to brown and then to pale pink with the naked eye as the pH values change from 9.0 to 1.0. Meanwhile, in Fig. S3 (b), the color of solutions varies from bright orange to green and then to nearly colorless with the decreases of the pH from 9.0 to 1.0. Moreover, the pH -sensitive feature of N-CDs was examined (Fig. 4a). The PL intensity of the N-CDs exhibited hardly obvious variation which was maximum at pH 7.0 when the pH ranges from 9.0 to 4.0. But it is evident that the PL intensity decreases sharply at lower pH (1.0–3.0) value. It may be due to the fact that amine groups are more likely to be protonated under strong acid conditions which possibly induces the aggregation of N-CDs, leading to the fluorescence quenching [47–50]. At the same time, the generation of new surface state (NH3+) induces slight blue shift of the PL peaks when the pH is decreasing [51]. To sum up, the N-CDs could be served as the dual-mode pH signal sensing from both fluorescence intensity and color change. It is sensible and convenient to exploit this property to detect pH by identifying the color of N-CDs solution. What's more, the fluorescent luminescence characteristic of N-CDs solution is easily affected by the extreme acidic (pH 1.0–3.0) conditions corresponding to the pH value of human gastric juice. It is expected to be applied to detect stomach diseases.
3.3. Sensitive and selective detection of Fe3+ It is also very important to detect Fe3+ in water solutions since Fe3+ ion exists in large quantities in the environment and exerts vital role in many physiological and pathological fields by combining with various regulatory proteins. In this research, the sensitivity of N-CDs for detecting Fe3+ was investigated. Fig. S4 displays two photographs of NCDs solutions (1 mg·mL−1) of different Fe3+ concentrations under visible and UV light illumination. The fluorescence color of N-CDs (Fig. S4.b) is gradually changing from bright orange to yellow to grass green. Additionally, the color of the N- CDs solutions under sunlight (Fig. S4.a) is from light orange to brown. Moreover, in Fig. 5a, the PL intensity of N-CDs gradually decreases with the increasing Fe3+ concentrations (0–300 μM), supported by gradual color variation of N-CDs optical photos under 365 nm light excitation. Furthermore, the fluorescent quenching tendency exhibited a fine linear relationship (R2 = 0.9912) as the concentration of Fe3+ is in the range of 0–200 μM. The selectivity performance of the N-CDs is furthermore explored. It is evident that the N-CDs exhibit sensitive selectivity toward Fe3+ ions over other metal ions like K+, Na+, Zn2+, Ni2+, Mn2+, Mg2+, Fe2+,
Fig. 4. (a) Fluorescence spectra of the N-CDs dispersed different pH values (1.0–9.0); (b) the reversible PL intensity of N-CDs dispersion when the pH value alternates between 2.0 and 7.0. 4
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Fig. 5. (a) Fluorescent emission spectra of the N-CDs in the presence of different concentrations of Fe3+from 0 to 300 μM. (b) The fluorescent responses to various metal ions.
Fig. 6. Sensing principle of nanoprobes for Fe3+.
Fig. 7. (a) Fluorescence decay of N-CDs in the absence of 200 μM Fe3+; (b) Fluorescence decay of N-CDs in the presence of 200 μM Fe3+.
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Cd2+, Ca2+, Ba2+ and Fe3+ metal ions. As exhibited in Fig. 5b, it's clearly illustrated that the other interference metal ions exhibited no evident effect on the fluorescence intensities of the N-CDs. However, once Fe3+ ion is added in the above testing solutions, the PL intensities of the N-CDs is significantly quenched. From the above results, it was proposed a possible mechanism to explain quenching mechanism of NCDs by Fe3+ ion (Fig. 6). The fluorescent quenching effect of N-CDs may result from the complexes of N-CDs and Fe3+ ion. It is known that the Fe3+ ion with half-filled 3d orbital could form the effective coordination with the various functional groups (e.g., hydroxyl, amino) on the surface of N-CD through strong interaction [52]. Especially, N atom on the surface of the N-CDs can serve as electron donors, which is also critical for increasing the local electronic density of the N-CDs. Such interactions facilitate the abundant electrons in the excited state of N-CDs transferring to the 3d orbital of Fe3+ and finally cause the strong fluorescence quenching. To further elucidate the detection mechanism, the PL lifetime is measured. As shown in Fig. 7, the PL lifetime of the N-CDs reduces from 7.14 ns to 6.09 ns when 200 μM Fe3+ ion was added. It suggests that the fluorescence quenching is dynamic, which clearly illustrates an ultrafast excitation recombination process.
fluorescence under 405 nm excitation wavelength which clearly indicates that the N-CDs own outstanding cell permeability. In the second investigation, HeLa cells incubating with the N-CDs/Fe3+ (200 μM) for 1.5 h (Fig. 8(d–f)) emits grass green fluorescence. These phenomena demonstrate that the N-CDs prepared could be applied for the cellular imaging with low cytotoxicity and certain water solubility. 4. Conclusion Overall, bright orange N-CDs were synthesized using the pPhenylendiamine as the precursor and ammonia as the nitrogen source by hydrothermal way. The as-prepared N-CDs exhibit excellent quantum yield (QY) (15.8%), abundant functional groups, outstanding biocompatibility and cell imaging ability. Such N-CDs can act as fluorescent nanosensors with multiple applications for detecting pH and Fe3+ ion selectively and sensitively. More importantly, the detection of pH values and Fe3+ was immediate by both visible and fluorescence variation. Furthermore, the N-CDs can be successfully introduced into HeLa cells to monitor intracellular Fe3+ on the basis of fluorescence intensity variation.
3.4. Biological application of N-CDs It is well known that excess and scarcity of Fe3+ could influence the cellular homeostasis, leading to various diseases. Thus, it's essential to further evaluate the feasibility of the N-CDs for imaging in living cells by the laser confocal microscope. The Fig. 8(a–c) represents the living image of HeLa cells cultivating with N-CDs (1.0 mg/ mL) for 1.5 h under corresponding bright-field, fluorescence and merged. As shown in the Fig. 8(a–c), the confocal microscopic images show bright orange
Acknowledgments We sincerely express gratitude to the Fundamental Research Funds for the Central Universities of China (2242016K41018 and KYLX16_0193) and A Project subsidized the Priority Academic Program Development of Jiangsu Higher Education Institutions (1107047002).
Fig. 8. Fluorescence microscopyimages for HeLa cells. (a–c) HeLacells incubated with the probe N-CDs (50 μg mL−1) for 1.5 h. (d–f) The cells were incubated with NCDs/Fe3+ (200 μM) for1.0 h.
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Appendix A. Supplementary data
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