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Nitrogen and sulfur co-doped highly luminescent carbon dots for sensitive detection of Cd (II) ions and living cell imaging applications
Journal of Photochemistry & Photobiology, B: Biology 186 (2018) 144–151
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Nitrogen and sulfur co-doped highly luminescent carbon dots for sensitive detection of Cd (II) ions and living cell imaging applications Dan Gu, Liu Hong, Lei Zhang, Hao Liu, Shaoming Shang
T
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Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Scallion Carbon dots Cd2+ Cell imaging
In this work, we have developed a green, simple and fast one-pot microwave-assisted strategy for synthesis of nitrogen and sulfur co-doped fluorescent carbon dots (CDs) using scallion (SL) as the carbon source. Optical properties of the SL-CDs have been measured by UV-visible and fluorescent spectroscopy. The morphology of the prepared SL-CDs has been performed by transmission electron microscopy (TEM). Surface functionality and elemental composition of SL-CDs was analyzed by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) spectra. The photoluminescent (PL) quantum yield of the obtained scallion carbon dots (SL-CDs) can reach as high as 18.6%. We further demonstrated that the SL-CDs can be used as fluorescent probes for detection of Cd2+ ions with a high sensitivity and an excellent selectivity. Linear relationships between the variation of the luminescent intensity of the SL-CDs before and after exposing the Cd2+ ions versus the concentration of Cd2+ ions in the range of 0.1–3.0 μM and 5.0–30.0 μM. The detection limit of Cd2+ ions can reach 15.0 nM. Moreover, the as-prepared SL-CDs exhibit negligible or extremely low cytotoxicity, which makes them be able to be used as fluorescent probes for living cell imaging. Overall, the prepared SL-CDs have promising applications in sensing of Cd2+ ions and in vivo or in vitro bioimaging.
1. Introduction Pollution induced by various heavy metal ions has become a critical worldwide issue threatening the health of human beings and the ecosystem. Among miscellaneous heavy metal ions, Cd2+ has been proven to be a highly toxic heavy metal ion, which is widely applicated in weapons industry, metallurgy electroplating and agriculture [1–4]. Serious injury to the lung, kidney, bone, nervous system, and even certain cancers will be caused to those people continuously exposed to even a minute amount of Cd2+ ions through the englobement of polluted food or water [5–7]. Therefore, sensitive detection of Cd2+ is highly desired. Well-known detection approaches for Cd2+ ions include atomic absorption spectrometry [8], inductively coupled plasma mass spectrometry (ICP-MS) [9], spectrophotometric method [10], and stripping voltammetry. Although these methods have high sensitivity and multiplex detection capability, the complicated sample preparation procedures and the high cost and time-consuming detection process prohibits their applications in many real cases [11]. Therefore, developing simple methods that can selectively and sensitively detect Cd2+ ions is urgent. The fluorescent sensor has attracted extensive attention in recent years due to its merits including simplicity, cost effectiveness, high sensitivity, intuitiveness, and fast response. So far, diverse ⁎
fluorescent probes have been developed relying on organic dye molecules, metal nanoparticles and semiconductor quantum dots (QDs). However, most of the above probes are usually either toxic or with low sensitivity and poor selectivity. Moreover, sensitive and highly selective detection of Cd2+ ions are troublesome since they have very similar chemical and physical properties to Zn2+ ions [12, 13]. Up to now, only a few highly selective fluorescence chemical sensor have been developed for Cd2+ detection [14–15]. So, it is still a challenge to explore new sensor for high selectivity and sensitivity towards Cd2+ against Zn2+ in aqueous solution. After more than ten years' development, carbon dots (CDs) have grown to be an extraordinarily bright fluorescent probes with many unique advantages including good solubility in aqueous solutions, low toxicity, outstanding biocompatibility, and environmental friendliness, as well as good sensitivity and selectivity, when compared with organic dyes and traditional semiconductor quantum dots [16–19]. Moreover, carbon dots have been applied in a wide range of fields ranging from bioimaging [20–22] to catalysis [23] and sensor [24]. Previous studies have proved that not only surface functionalization/passivation, but heteroatom doping can improve the performance of CDs in the abovementioned applications. Nowadays, many efforts have been focused on controllable
Corresponding author. E-mail address: [email protected] (S. Shang).
https://doi.org/10.1016/j.jphotobiol.2018.07.012 Received 16 January 2018; Received in revised form 13 July 2018; Accepted 17 July 2018 Available online 18 July 2018 1011-1344/ © 2018 Elsevier B.V. All rights reserved.
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large particles. The resultant transparent supernatant was collected and filtered using a polytetrafluoroethylene (PTFE) syringe filter with 0.22 μm pore size on average. By performing the filtration step, a darkbrown solution was obtained. The dark-brown solution was dialyzed in deionized water though a dialysis membrane (i.e., 1000MWCO) for 3 days. The deionized water was changed for every 4 h. Eventually, high quality carbon dots were fabricated. Dry SL-CDs were obtained by lyophilizing the transparent and brown aqueous solution.
synthesis of CDs doped with heteroatom, especially those doped with nitrogen and sulfur atoms with designable fluorescent properties [25–27]. Biomass has a plenty of carbohydrates that can supply a large amount of carbon atoms for the preparation of CDs. Meanwhile, the biomass usually contains different proteins and other molecules that can provide heteroatoms including N and S elements. Therefore, biomass has been used to prepare CDs such as bee pollens [28], hair [29], banana juice [30], winter melon [31] and potato [32]. Here, considering plentiful carbon, nitrogen, oxygen and sulfur elements existed in the proteins of scallion (SL), other than the vitamin, amino acid, carbohydrates, niacin, and lipid, we explored green fabrication of CDs doped with N and S atoms with controllable luminescent properties using scallion as a precursor. More importantly, the synthetic N and S co-doped SL-CDs were used to differentiate Cd2+ ions from other ions including Zn2+ with a high sensitivity. Moreover, these SLCDs were employed in bioimaging applications benefited from their strong luminescence and biocompatibility. Overall, we developed a one-step microwave-assisted synthetic method to prepare N and S codoped CDs with controllable and strong luminescence using scallion as a precursor. These SL-CDs can be used to detect and differentiate Cd2+ ions with a high sensitivity and also be adopted in bioimaging applications.
2.3. Quantum Yield Evaluation of the Photoluminescence from SL-CDs The quantum yield (QY) of the photoluminescence from the SL-CDs was determined according to a previously reported procedure with quinine sulfate (whose QY is 54% [33] when dissolved in 0.1 M H2SO4) as a reference. To suppress the reabsorption effects, absorbance in the fluorescence cuvette of 10 mm thickness were maintained smaller than 0.1 at the excitation wavelength of 360 nm [34]. The following equation was used to evaluate the QY of the SL-CDs:
φx = φre × (Ix / Ire ) × (Are / Ax ) × (n x / nre )2 . where φ indicates the QY, I suggest the calculated integrated fluorescent emission intensity, A denotes the optical intensity at excitation wavelength, n implies the refractive index of the solvent. The subscript “x” refers to the sample and “re” refers to the reference with predetermined QY. In our case, nx = 1.33 and nre = 1.33.
2. Experimental 2.1. Materials and Characterization Scallions were bought from a local supermarket and washed several times with deionized water before usage. HgCl2, BaCl2.2H2O, CuCl2, Pb (NO3)2, FeCl3.6H2O, CoCl2.6H2O, FeSO4.7H2O, ZnSO4.7H2O, CaCl2, MgCl2.6H2O, Al2(SO4)3.18H2O, 3CdSO4.8H2O, CsCl, HCl, NaOH, and NaCl were purchased from Sinopharm Chemical Reagent Co.,Ltd., (Shanghai, China), which are used as received without further purification. The standard solutions of Cd2+ were purchased from National Center of Analysis and Testing for Nonferrous Metalsand Electronic Materials (Beijing, China). Deionized water was used in all experiments. Every chemical possessed the analytical quality and was utilized with no additional purification. The morphology and structure of the SL-CDs were observed by a JEOL JEM-2100 high-resolution transmission electron microscope (Tokyo, Japan) with an accelerating voltage of 200 kV. The fluorescence spectra of the SL-CDs were recorded in a quartz cuvette (10 mm × 10 mm) on a Varian Cary Eclipse spectrofluorometer (Palo Alto, CA, USA) with excitation and emission slit width at 5 nm. UV–Visible absorption spectrum was measured by using a TU-1901 UV–Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., china). FTIR spectrum was performed on a FALA2000104 spectrophotometer. The fluorescence lifetime was measured using a Delta flex UltraFast lifetime spectrofluorometer (Horiba Jobinyvon IBH Inc., UK). X-ray photoelectron spectroscopy (XPS) was obtained using a Kratos AXIS Ultra DLD spectrometer (Shimadzu, Japan) equipped with a mono X-Ray source Al Kα excitation (1486.6 eV). The reaction was carried out in a G80F23CN2P-B5 (R0) microwave oven (Galanz Microwave Oven Co., Ltd., China) with power of 800 W. The size distribution of the as-prepared SL-CDs was revealed by dynamic light scattering (DLS) measurements using a Nano ZS90 instrument (Malvern).
2.4. Detection of Cd2+ Ions Using SL-CDs Typically, 10 μL of SL-CDs dispersion was introduced into acetate buffer solutions (pH = 5). Then, Cd2+ ions were introduced into the above solution at different concentrations. The photoluminescent (PL) spectra of the solution contained Cd2+ ions were recorded after the solution stored for 3.0 mins at room temperature. The detection selectivity of SL-CDs in detecting Cd2+ ions was evaluated by measuring the PL spectra of the solution contained different control ions. The fluorescent intensities from the SL-CDs in the inexistence (F0) or existence (F) of the control ions were measured. The selectivity and the sensitivity in sensing Cd2+ ions were measured for three times and the average values were presented for accuracy.
2.5. Cellular Toxicity Test and Cell Imaging Applications The MTT assay method was used to study the cytotoxicity of the SLCDs to A549 cells. Human lung adenocarcinoma A549 cells were first seeded into a plate with 96 wells at a concentration of about 2 × 103 cells per well and then cultured at 37 °C for 24 h under a 5% CO2 atmosphere. In consequence, different amounts of SL-CDs were introduced into each well and incubated for another 24 h. Consequently, 10 μL of MTT solutions (at a concentration of about 5 mg/mL in a phosphate buffered saline solution) were added to each well and incubated for 4 h at 37 °C. The culture medium was removed before pipetting 100 μL of DMSO to each well followed by shaking for 10 mins. At last, a microplate reader was used to measure the optical density of the mixture at 570 nm wavelength. In the propagation period, the A549 cells were dispersed in 24 replicate wells and incubated at 37 °C for 24 h in an incubator filled with 5% CO2. The culture medium was replaced by a fresh medium containing SL-CDs at a concentration of 50 μg/mL. The A549 cells were incubated for another 2 h in the medium composed of SL-CDs. The A549 cells were washed three times with a PBS buffer solution before imaging with an inverted Olympus IX51 fluorescence microscope.
2.2. Preparation of SL-CDs In a typical synthesis, 10.0 g of freshly chopped scallions was introduced into a beaker and then transferred to a domestic 800 W microwave oven. Then the oven was heated for 4 min. Subsequently, 50 mL of deionized water was poured into the reaction mixture after it cooled to room temperature naturally. In consequence, the obtained turbid liquid was centrifuged at 8000 rpm for 10 min to remove those 145
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are ascribed to C-N-C and surface H2N- groups, respectively. The XPS results further verified that the nitrogen and sulfur atoms have been successfully doped into the SL-CDs. Two peaks located at 532.8 eV and 531.9 eV were observed in the O1s XPS spectrum, arising from the COH and C-N-O groups, respectively (Fig. 3e). Three peaks centered at 163.4 eV, 164.5 eV, 167.8 eV, emerged in the S2p XPS spectrum (Fig. 3f), underlying the existence of three forms of sulfur element. The -C-S- (S2p, 3/2 and S2p, 1/2) covalent bond of the thiophene-S owing to the spin-orbit couplings gives rise to the formation of the former two peaks [35]. The -C-SO3- species, such as sulfate or sulfonate, induce the emergence of the last peak [29]. The above FTIR and XPS results indicated that the synthetic SL-CDs are functionalized by those groups containing oxygen, nitrogen and sulfur elements. These functional groups make the SL-CDs have excellent water solubility and facilitate their further desired modifications towards different application purposes.
Fig. 1. Scheme of the synthesis process of SL-CDs and the picture of the corresponding sample under 365 nm ultraviolet lamp illumination.
3. Results and Discussion 3.1. Characterization of SL-CDs
3.2. Optical Properties
As exhibited in Fig. 1, The SL-CDs were preparated using scallion as the carbon source via a simple microwave treatment. The size and morphology of the SL-CDs were performed by the transmission electron microscopy (TEM) (Fig. 2a). The TEM characterization demonstrated that the well-dispersed SL-CDs are spherical particles and the average diameter is 3.23 nm (Fig. 2b). Dynamic light scattering results (Fig.S1) suggested that the average diameter of SL-CDs is approximately 6.36 nm. The diameter measured from DLS characterization is slightly larger than that obtained from TEM characterization because the former represents the hydrodynamic diameter of SL-CDs. X-ray photoelectron spectroscopy (XPS) and Fourier. Transformed infrared (FTIR) absorption were measured to investigate the composition and chemical structure of the SL-CDs. As seen in the FTIR spectra (Fig. 3a), the peak centered at 3284 cm−1 is arising from the stretching vibration of –OH and H2N- groups. The stretching vibration of CeH bonds give rise to the appearance of the band at 2930 cm−1. The peak located at 1596 cm−1 originates from the bending vibrations of NeH. The 1401 cm−1 peak is contributed by the CeN stretching vibrations. The peak situated at 1245 cm−1 is identified to be the CeS stretching vibrations. The CeO stretching vibrations generate the peak at 1026 cm−1. The XPS spectrums provide rich information about the surface functionalization stage of SL-CDs. The XPS survey spectrum of the SL-CDs exhibited four prominent peaks originating from C1s, O1s, N1s, and S2P (Fig. 3b). The percentage of C, O, N, and S is 57.63%, 37.41%, 3.98% and 0.98%, respectively. The C1s XPS spectrum revealed that there are four different types of carbon atoms (Fig. 3c), that is, C=C/C-C with a binding energy at 284.5 eV, CeO at 286.4 eV, C]O at 287.9 eV, and C-N/C-S at 285.2 eV. The N1 s XPS spectrum showed two peaks at 400.1 eV and 401.1 eV (Fig. 3d), which
The prominent absorption peak located at 280 nm in the UV–Vis absorption spectrum is ascribed to the π-π* transition of C]C [36]. The PL spectrum of SL-CDs showed a strong peak at 455 nm when the excitation wavelength is 390 nm. The diluted solution of the SL-CDs is light yellow colour under sun light, while turns to blue under 365 nm ultraviolet lamp illumination (Fig. 4a). The PL spectrum of the SL-CDs showed obvious excitation wavelength-dependent property, as usually observed by previous publications for fluorescent carbon materials. This excitation wavelength-dependent property is caused by the fact that the surface state of the SL-CDs can affect the band gap of SL-CDs [37]. The surface state of the SL-CDs is analogous to a. Molecular state; while the size effect is induced by the quantum confinement effect when the size of the SL-CDs is pretty small. The surface state and the size effect coordinate together to endow the complex PL properties of the SL-CDs [38]. The PL emission peaks are red-shifted from 425 nm to 525 nm as the excitation wavelength changed from 340 nm to 470 nm; The PL intensity also changed (Fig. 4b). These PL properties of the SL-CDs make the CDs suitable to be used in optical bioimaging in vitro and in vivo, especially after considering the formative needs for molecular probes in cellular imaging [39]. The PL QY of SL-CDs was calculated to be approximately 18.6% at the excitation wavelength of 360 nm by using the PL QY of quinine sulfate as reference. The measured absorbance of SL-CDs and quinine sulfate at 360 nm for QY calculation were 0.026 and 0.011, respectively. The corresponding integrated fluorescent emission intensities were 4095 and 5030, respectively. The corresponding fluorescence emission spectra under the excitation wavelength of 360 nm are depicted in Fig.S2. This QY value of the SL-CDs prepared in our work is
Fig. 2. a Transmission electron microscopy images of SL-CDs, b Particle diameter distribution of SL-CDs. 146
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Fig. 3. a The FT-IR spectra of the SL-CDs, The XPS (b), C1s (c), N1 s (d), O1s (e) and S2p (f) spectra of the as-attained SL-CDs.
Fig. 4. a UV–Vis absorption and PL emission spectra of the aqueous solution of the SL-CDs (Insets show the photographs of SL-CDs under visible light (left) and UV light (right), b PL spectra of the SL-CDs at varied excitation wavelength ranging from 340 to 470 nm.
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ions over other competitive cations including Zn2+ ions. To evaluate the detection limit for Cd2+ detection, the medium conditions were further optimized. The influence of pH value of the medium on the detection sensitivity of Cd2+ ions was investigated (Fig. S6a). We found that the optimum quenching efficiency was achieved when the pH value of the solution is around 5.0. To find the best buffer solution, the effects of four different buffer solutions with a pH value of 5.0 were further studied (Fig. S6b). Among them, HAc-NaAc (ACE) buffer solution was found to be the best buffer medium as the highest △F is obtained when ACE is used. Therefore, the ACE buffer solution with a pH value of 5.0 was used in this work. The ratio of F/F0 keeps constant when the reaction time is ≥180 s (Fig. S6c), demonstrating that the detection of Cd2+ ions using SL-CDs only needs a short time. 180 s was chosen as the reaction time for further studies. As expected, the PL intensity is reduced gradually as the concentration of Cd2+ ions increases (Fig. 6a), implying that the PL intensity of the SL-CDs is very sensitive to Cd2+ ions. The relationship between the concentration of Cd2+ ions and the ratio of F/F0 follows two lines in different concentration ranges of Cd2+ ions (Fig. 6c and d). When the concentration of Cd2+ ions is in the range of 0.1–3.0 μM, the relationship can be described by F/F0 = 1.00084–0.15702 × [Cd2+] (R2 = 0.98586), and the corresponding binding constant is 2.816 × 105 ± 11,670 L mol−1. When the concentration is in the range of 5.0–30.0 μM, the relationship is F/F0 = 0.51008–0.00588 [Cd2+] (R2 = 0.99397), and the corresponding binding constant is 3.636 × 104 ± 2120 L mol−1. The detection limit of Cd2+ ions can reach as low as 15 nM at a signal-to-noise ratio (S/N) of 3. The obtained detection limit is comparable to previously reported sensing methods in the literature (Table S2). To investigate the potential of our detection method in real cases, we studied analysis of the tap water samples. As shown in Table 1, it can be seen that the results of recovery for the samples are satisfied.
higher than those of CDs reported previously (Table S1), which demonstrates the extraordinary fluorescent properties of the SL-CDs [40]. The high PL quantum yield of the SL-CDs is beneficial to the applications in cell imaging and detection Cd2+ ions as shown in the following content. The PL properties of the SL-CDs under different pH values were further studied (Fig. S3a). The PL intensity of the SL-CDs increases as the pH value was increased from 2 to 3, while kept stable as the pH value was further increased from 3 to 9. When the pH was larger than 9, the PL intensity gradually reduced. This means that the SL-CDs can be used as a pH sensor to monitor the pH distribution within cells. The effects of pH on the absorption spectral changes are shown in Fig.S3b. The absorption peak showed no obvious change. The absorbance value of the SL-CDs increases as the pH value was increased from 2 to 3, while kept stable as the pH value was further increased from 3 to 9. When the pH was larger than 9, the absorbance value gradually reduced. The PL intensity did not show significant change when the solution containing different amounts of NaCl (Fig. S3c). More importantly, the PL intensity of the SL-CDs did not reduce when continuously irradiated under a 365 nm UV-lamp with the distance of 15 cm between the sample and light source (Fig. S3d), demonstrating the fascinating photostability of the fluorescence probe. The photostability of the SL-CDs makes them promising for application in bioimaging.
3.3. SL-CDs Nanoprobe for Cd2+ Detection To thoroughly evaluate the selectivity of the SL-CDs in detecting different metal ions, the PL intensity variations before and after introducing 50 μM of metal ions, including Cs+, Cd2+, Hg2+, Cu2+, Pb2+, Al3+, Fe2+, Zn2+, Ba2+, Mg2+, Ca2+, Co2+, and Fe3+ into the SL-CDs solution were investigated separately. As shown in Fig. 5a and Fig.S4, the PL of the SL-CDs is dramatically quenched after. The addition of Cd2+ ions. In a sharp contrast, no or very unobvious PL quenching phenomenon is observed when other metal ions are introduced into the colloidal solution of SL-CDs. This means that the SLCDs can be used as an efficient fluorescence probe for Cd2+ detection. The obvious fluorescence quenching of the SL-CDs after Cd2+ addition may be owing to the special binding between the Cd2+ ions and the phenolic hydroxyl and/or amine groups of SL-CDs. Moreover, we measured the fluorescence intensity of SL-CDs in the presence of CdCl2, Cd(NO3)2, and Cd(SO4)2 at the same concentration to eliminate the effects of Cd2+ counter ions (Fig.S5). The obtained results indicate almost the same PL quenching phenomenon. Thus, the changes were not due to the specific counter ion of Cd2+. The interference test results were shown in Fig. 5b, demonstrating that the other interference metal ions showed no obvious effect on the PL intensities of the SL-CDs. Nevertheless, once Cd2+ ions are introduced into the testing solutions, significant fluorescence quenching is observed. These results confirm that the SL-CDs have an excellent selectivity during detection of Cd2+
3.4. Possible Mechanism of the Fluorescence Response of SL-CDs to Cd2+ To deepen our understanding about the PL quenching mechanism of. SL-CDs by Cd2+, UV–Vis absorption spectra, the effects of temperatures on quenching reaction, the lifetime of the PL and the quenching rate constant Kq were recorded. As shown in Fig.7a, the absorption peak of pure SL-CDs is centered at 280 nm. Nevertheless, this absorption peak gets weakened significantly when Cd2+ was introduced into the colloidal solution of SL-CDs, whereas no obvious absorption spectral changes were observed with the addition of other metal cations (Fig.S7). We conclude that this process is a static quenching process, because dynamic quenching only affects the excited state of fluorescent molecules and has no effect on ultraviolet absorption [41]. And the hydroxyl at surface of SL-CDs can react with metal ions, thus can infer a non-luminous ground state compounds were Fig. 5. a Fluorescent response of SL-CDs to different metal ions in HAc-NaAc buffer solution (pH 5.0). 50 μmol/L is used for each metal ion. F0 and F represent the fluorescent intensity of SL-CDs at λem of 455 nm excited at 390 nm in the inexistence and existence of metal ions, respectively, b The fluorescent intensity of SL-CDs (red) and SL-CDs + Cd2+ (blue) solutions in the existence of various metal ions (50 μmol/L). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. a PL emission spectra of SL-CDs upon addition of various concentrations of Cd2+ in HAc-NaAc (pH = 5) at an excitation wavelength of 390 nm. Inset: Plot of the fluorescent intensity against Cd(II) concentration within the range of 0–50 μM, b Sensing principle of the SL-CDs based probe for Cd2+, The linear relationship of F/F0 against the Cd2+ concentration over the range of (c: 0.1–3.0 μM; d: 5.0–30.0 μM).
molecular motion accelerates. The result will increase the diffusion coefficient of the molecule. Finally, the degree of quenching is increased. On the contrary, the stability of the complex may decrease as the temperature is increased, so the degree of static quenching is reduced. According to these, the quenching of Cd2+ on SL-CDs should be static quenching, this is consistent with the results of ultraviolet absorption. The interaction of quenching agent and the excited state of fluorescence substance leads to dynamic quenching, and the interaction with the ground state of fluorescence substance leads to static quenching [41]. The dynamic quenching process is the process of competing with spontaneous emission process to shorten the lifetime of excited molecules. For static quenching, the existence of quenching agent will not change the lifetime of excited fluorescence molecules. The fluorescent lifetime of SL-CDs is determined to be 3.43 ns, which turns to be 3.41 ns after the addition of Cd2+ ions (Fig.7b). The slightly reduced fluorescent lifetime demonstrates that the mechanism of Cd2+ quenching of SL-CDs is a static quenching. So, compared with other metal ions, Cd2+ ions have more cogent affinity and faster chelating kinetics towards the hydroxyl on the surface of the SL-CDs.
Table 1 Detection of Cd2+ in Tap water. Sample
Added (μmol/L)
Found (μmol/L)
Recovery (%, n = 5)
Tap water
0.2 0.5 2.5 10 20
0.188 ± 0.008 0.489 ± 0.007 2.575 ± 0.009 9.9 ± 0.008 21.2 ± 0.009
94% 97.8% 103% 99% 106%
formed by Cd2+ with hydroxyl on the surface of SL-CDs. So, the ultraviolet absorption and fluorescence intensity of SL-CDs were reduced. As shown in Fig.S8, the degree of quenching decreases as the temperature was increased from 15 °C to 50 °C. However, the quenching phenomenon is not obvious when the concentration of Cd2+ is low, indicating that Cd2+ and SL-CDs formed a nonluminous complex. More and more nonluminous complex are formed as the Cd2+ concentration was increased. So, the quenching becomes more and more obvious. The dynamic quenching is related to diffusion coefficient. The viscosity of the solution decreased when the temperature was increased, and the
Fig. 7. a UV–Vis absorption spectra of the Cd2+ ions (black line), SL-CDs (blue line) and SL-CDs after exposing to Cd2+ ions (red line). b Fluorescent decay traces of the SL-CDs in the inexistence (black line) and existence (red line) of Cd2+ ions, (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 8. Confocal fluorescent pictures of A549 cells incubated with SL-CDs for 24 h under (a) 405 nm excitation and (b) bright field, (c) The merged image of a and b.
In order to further study the quenching process between Cu2+ and carbon dots, The quenching rate constant Kq is calculated by Kq = Ksv/ τ0, wherein, Ksv is Stern-Volmer quenching constant, which is the binding constant of the complex formed by quencher and fluorescent substance for static quenching. τ0 is the fluorescence lifetime of carbon dots without quenching agent. The calculated value of Kq is 8.2 × 1013 L/(mol·s), which is 1000 times higher than the upper limit of the typical dynamic quenching rate constant 1.0 × 1010 L/(mol·s), indicating that the quenching of carbon dots by Cd2+ is caused by the formation of the non-luminescent complex, which is static quenching process.
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3.5. Cytotoxicity and Cellular Imaging The cytotoxicity of luminescent quantum dots is important for living cell imaging applications. Therefore, to explore the potential application of SL-CDs in living cell imaging, the cytotoxicity of SL-CDs is first evaluated by the MTT assay in A549 cells. The A549 cell viabilities were evaluated after the cells are exposed to SL-CDs at different concentrations. The SL-CDs have negligible or extremely low cytotoxicity (Fig. S9). Almost 100% of the cells retain viable even after exposing SLCDs at a concentration of up to 10 μg/mL. There are still 92% of the cells viable after exposing SL-CDs at a concentration as high as 50 μg/ mL. Therefore, the SL-CDs are safe for in vitro and in vivo cell imaging applications. Moreover, a prominent intracellular fluorescence is observed after the SL-CDs incubated with A549 cells. Fig. 8 displayed the samples observed under bright field and excited at 405 nm by a fluorescence microscopy. The result demonstrated that the fluorescent signals are from the perinuclear regions of the cytosol, indicating the outstanding cell-permeability of the SL-CDs into living cells owing to their beneficial surface functional groups and small size. 4. Conclusion In summary, a facile method was developed to synthesize N- and S co-doped carbon dots using scallion as the carbon source. The obtained SL-CDs exhibit desired fluorescent properties with a PL QY of 18.6%, which makes them be able to serve as an effective fluorescence probe for sensitive detection of Cd2+ ions. The detection limit can reach as low as 15 nM. Detection of Cd2+ ions in environmental water demonstrated this sensor can be successfully used to monitor the concentration of Cd2+ ions in daily used water. Eventually, the low toxicity and strongly fluorescent carbon dots were applied for cell imaging and the quenched fluorescence by adding Cd2+, demonstrating their potential towards diverse applications. Acknowledgements The authors would like to thank a Grant from the Natural Science Foundation of Jiangsu Province (BK20170175). 150
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Nitrogen and sulfur co-doped highly luminescent carbon dots for sensitive detection of Cd (II) ions and living cell imaging applications Dan Gu, Liu Hong, Lei Zhang, Hao Liu, Shaoming Shang
T
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Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Scallion Carbon dots Cd2+ Cell imaging
In this work, we have developed a green, simple and fast one-pot microwave-assisted strategy for synthesis of nitrogen and sulfur co-doped fluorescent carbon dots (CDs) using scallion (SL) as the carbon source. Optical properties of the SL-CDs have been measured by UV-visible and fluorescent spectroscopy. The morphology of the prepared SL-CDs has been performed by transmission electron microscopy (TEM). Surface functionality and elemental composition of SL-CDs was analyzed by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) spectra. The photoluminescent (PL) quantum yield of the obtained scallion carbon dots (SL-CDs) can reach as high as 18.6%. We further demonstrated that the SL-CDs can be used as fluorescent probes for detection of Cd2+ ions with a high sensitivity and an excellent selectivity. Linear relationships between the variation of the luminescent intensity of the SL-CDs before and after exposing the Cd2+ ions versus the concentration of Cd2+ ions in the range of 0.1–3.0 μM and 5.0–30.0 μM. The detection limit of Cd2+ ions can reach 15.0 nM. Moreover, the as-prepared SL-CDs exhibit negligible or extremely low cytotoxicity, which makes them be able to be used as fluorescent probes for living cell imaging. Overall, the prepared SL-CDs have promising applications in sensing of Cd2+ ions and in vivo or in vitro bioimaging.
1. Introduction Pollution induced by various heavy metal ions has become a critical worldwide issue threatening the health of human beings and the ecosystem. Among miscellaneous heavy metal ions, Cd2+ has been proven to be a highly toxic heavy metal ion, which is widely applicated in weapons industry, metallurgy electroplating and agriculture [1–4]. Serious injury to the lung, kidney, bone, nervous system, and even certain cancers will be caused to those people continuously exposed to even a minute amount of Cd2+ ions through the englobement of polluted food or water [5–7]. Therefore, sensitive detection of Cd2+ is highly desired. Well-known detection approaches for Cd2+ ions include atomic absorption spectrometry [8], inductively coupled plasma mass spectrometry (ICP-MS) [9], spectrophotometric method [10], and stripping voltammetry. Although these methods have high sensitivity and multiplex detection capability, the complicated sample preparation procedures and the high cost and time-consuming detection process prohibits their applications in many real cases [11]. Therefore, developing simple methods that can selectively and sensitively detect Cd2+ ions is urgent. The fluorescent sensor has attracted extensive attention in recent years due to its merits including simplicity, cost effectiveness, high sensitivity, intuitiveness, and fast response. So far, diverse ⁎
fluorescent probes have been developed relying on organic dye molecules, metal nanoparticles and semiconductor quantum dots (QDs). However, most of the above probes are usually either toxic or with low sensitivity and poor selectivity. Moreover, sensitive and highly selective detection of Cd2+ ions are troublesome since they have very similar chemical and physical properties to Zn2+ ions [12, 13]. Up to now, only a few highly selective fluorescence chemical sensor have been developed for Cd2+ detection [14–15]. So, it is still a challenge to explore new sensor for high selectivity and sensitivity towards Cd2+ against Zn2+ in aqueous solution. After more than ten years' development, carbon dots (CDs) have grown to be an extraordinarily bright fluorescent probes with many unique advantages including good solubility in aqueous solutions, low toxicity, outstanding biocompatibility, and environmental friendliness, as well as good sensitivity and selectivity, when compared with organic dyes and traditional semiconductor quantum dots [16–19]. Moreover, carbon dots have been applied in a wide range of fields ranging from bioimaging [20–22] to catalysis [23] and sensor [24]. Previous studies have proved that not only surface functionalization/passivation, but heteroatom doping can improve the performance of CDs in the abovementioned applications. Nowadays, many efforts have been focused on controllable
Corresponding author. E-mail address: [email protected] (S. Shang).
https://doi.org/10.1016/j.jphotobiol.2018.07.012 Received 16 January 2018; Received in revised form 13 July 2018; Accepted 17 July 2018 Available online 18 July 2018 1011-1344/ © 2018 Elsevier B.V. All rights reserved.
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large particles. The resultant transparent supernatant was collected and filtered using a polytetrafluoroethylene (PTFE) syringe filter with 0.22 μm pore size on average. By performing the filtration step, a darkbrown solution was obtained. The dark-brown solution was dialyzed in deionized water though a dialysis membrane (i.e., 1000MWCO) for 3 days. The deionized water was changed for every 4 h. Eventually, high quality carbon dots were fabricated. Dry SL-CDs were obtained by lyophilizing the transparent and brown aqueous solution.
synthesis of CDs doped with heteroatom, especially those doped with nitrogen and sulfur atoms with designable fluorescent properties [25–27]. Biomass has a plenty of carbohydrates that can supply a large amount of carbon atoms for the preparation of CDs. Meanwhile, the biomass usually contains different proteins and other molecules that can provide heteroatoms including N and S elements. Therefore, biomass has been used to prepare CDs such as bee pollens [28], hair [29], banana juice [30], winter melon [31] and potato [32]. Here, considering plentiful carbon, nitrogen, oxygen and sulfur elements existed in the proteins of scallion (SL), other than the vitamin, amino acid, carbohydrates, niacin, and lipid, we explored green fabrication of CDs doped with N and S atoms with controllable luminescent properties using scallion as a precursor. More importantly, the synthetic N and S co-doped SL-CDs were used to differentiate Cd2+ ions from other ions including Zn2+ with a high sensitivity. Moreover, these SLCDs were employed in bioimaging applications benefited from their strong luminescence and biocompatibility. Overall, we developed a one-step microwave-assisted synthetic method to prepare N and S codoped CDs with controllable and strong luminescence using scallion as a precursor. These SL-CDs can be used to detect and differentiate Cd2+ ions with a high sensitivity and also be adopted in bioimaging applications.
2.3. Quantum Yield Evaluation of the Photoluminescence from SL-CDs The quantum yield (QY) of the photoluminescence from the SL-CDs was determined according to a previously reported procedure with quinine sulfate (whose QY is 54% [33] when dissolved in 0.1 M H2SO4) as a reference. To suppress the reabsorption effects, absorbance in the fluorescence cuvette of 10 mm thickness were maintained smaller than 0.1 at the excitation wavelength of 360 nm [34]. The following equation was used to evaluate the QY of the SL-CDs:
φx = φre × (Ix / Ire ) × (Are / Ax ) × (n x / nre )2 . where φ indicates the QY, I suggest the calculated integrated fluorescent emission intensity, A denotes the optical intensity at excitation wavelength, n implies the refractive index of the solvent. The subscript “x” refers to the sample and “re” refers to the reference with predetermined QY. In our case, nx = 1.33 and nre = 1.33.
2. Experimental 2.1. Materials and Characterization Scallions were bought from a local supermarket and washed several times with deionized water before usage. HgCl2, BaCl2.2H2O, CuCl2, Pb (NO3)2, FeCl3.6H2O, CoCl2.6H2O, FeSO4.7H2O, ZnSO4.7H2O, CaCl2, MgCl2.6H2O, Al2(SO4)3.18H2O, 3CdSO4.8H2O, CsCl, HCl, NaOH, and NaCl were purchased from Sinopharm Chemical Reagent Co.,Ltd., (Shanghai, China), which are used as received without further purification. The standard solutions of Cd2+ were purchased from National Center of Analysis and Testing for Nonferrous Metalsand Electronic Materials (Beijing, China). Deionized water was used in all experiments. Every chemical possessed the analytical quality and was utilized with no additional purification. The morphology and structure of the SL-CDs were observed by a JEOL JEM-2100 high-resolution transmission electron microscope (Tokyo, Japan) with an accelerating voltage of 200 kV. The fluorescence spectra of the SL-CDs were recorded in a quartz cuvette (10 mm × 10 mm) on a Varian Cary Eclipse spectrofluorometer (Palo Alto, CA, USA) with excitation and emission slit width at 5 nm. UV–Visible absorption spectrum was measured by using a TU-1901 UV–Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., china). FTIR spectrum was performed on a FALA2000104 spectrophotometer. The fluorescence lifetime was measured using a Delta flex UltraFast lifetime spectrofluorometer (Horiba Jobinyvon IBH Inc., UK). X-ray photoelectron spectroscopy (XPS) was obtained using a Kratos AXIS Ultra DLD spectrometer (Shimadzu, Japan) equipped with a mono X-Ray source Al Kα excitation (1486.6 eV). The reaction was carried out in a G80F23CN2P-B5 (R0) microwave oven (Galanz Microwave Oven Co., Ltd., China) with power of 800 W. The size distribution of the as-prepared SL-CDs was revealed by dynamic light scattering (DLS) measurements using a Nano ZS90 instrument (Malvern).
2.4. Detection of Cd2+ Ions Using SL-CDs Typically, 10 μL of SL-CDs dispersion was introduced into acetate buffer solutions (pH = 5). Then, Cd2+ ions were introduced into the above solution at different concentrations. The photoluminescent (PL) spectra of the solution contained Cd2+ ions were recorded after the solution stored for 3.0 mins at room temperature. The detection selectivity of SL-CDs in detecting Cd2+ ions was evaluated by measuring the PL spectra of the solution contained different control ions. The fluorescent intensities from the SL-CDs in the inexistence (F0) or existence (F) of the control ions were measured. The selectivity and the sensitivity in sensing Cd2+ ions were measured for three times and the average values were presented for accuracy.
2.5. Cellular Toxicity Test and Cell Imaging Applications The MTT assay method was used to study the cytotoxicity of the SLCDs to A549 cells. Human lung adenocarcinoma A549 cells were first seeded into a plate with 96 wells at a concentration of about 2 × 103 cells per well and then cultured at 37 °C for 24 h under a 5% CO2 atmosphere. In consequence, different amounts of SL-CDs were introduced into each well and incubated for another 24 h. Consequently, 10 μL of MTT solutions (at a concentration of about 5 mg/mL in a phosphate buffered saline solution) were added to each well and incubated for 4 h at 37 °C. The culture medium was removed before pipetting 100 μL of DMSO to each well followed by shaking for 10 mins. At last, a microplate reader was used to measure the optical density of the mixture at 570 nm wavelength. In the propagation period, the A549 cells were dispersed in 24 replicate wells and incubated at 37 °C for 24 h in an incubator filled with 5% CO2. The culture medium was replaced by a fresh medium containing SL-CDs at a concentration of 50 μg/mL. The A549 cells were incubated for another 2 h in the medium composed of SL-CDs. The A549 cells were washed three times with a PBS buffer solution before imaging with an inverted Olympus IX51 fluorescence microscope.
2.2. Preparation of SL-CDs In a typical synthesis, 10.0 g of freshly chopped scallions was introduced into a beaker and then transferred to a domestic 800 W microwave oven. Then the oven was heated for 4 min. Subsequently, 50 mL of deionized water was poured into the reaction mixture after it cooled to room temperature naturally. In consequence, the obtained turbid liquid was centrifuged at 8000 rpm for 10 min to remove those 145
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are ascribed to C-N-C and surface H2N- groups, respectively. The XPS results further verified that the nitrogen and sulfur atoms have been successfully doped into the SL-CDs. Two peaks located at 532.8 eV and 531.9 eV were observed in the O1s XPS spectrum, arising from the COH and C-N-O groups, respectively (Fig. 3e). Three peaks centered at 163.4 eV, 164.5 eV, 167.8 eV, emerged in the S2p XPS spectrum (Fig. 3f), underlying the existence of three forms of sulfur element. The -C-S- (S2p, 3/2 and S2p, 1/2) covalent bond of the thiophene-S owing to the spin-orbit couplings gives rise to the formation of the former two peaks [35]. The -C-SO3- species, such as sulfate or sulfonate, induce the emergence of the last peak [29]. The above FTIR and XPS results indicated that the synthetic SL-CDs are functionalized by those groups containing oxygen, nitrogen and sulfur elements. These functional groups make the SL-CDs have excellent water solubility and facilitate their further desired modifications towards different application purposes.
Fig. 1. Scheme of the synthesis process of SL-CDs and the picture of the corresponding sample under 365 nm ultraviolet lamp illumination.
3. Results and Discussion 3.1. Characterization of SL-CDs
3.2. Optical Properties
As exhibited in Fig. 1, The SL-CDs were preparated using scallion as the carbon source via a simple microwave treatment. The size and morphology of the SL-CDs were performed by the transmission electron microscopy (TEM) (Fig. 2a). The TEM characterization demonstrated that the well-dispersed SL-CDs are spherical particles and the average diameter is 3.23 nm (Fig. 2b). Dynamic light scattering results (Fig.S1) suggested that the average diameter of SL-CDs is approximately 6.36 nm. The diameter measured from DLS characterization is slightly larger than that obtained from TEM characterization because the former represents the hydrodynamic diameter of SL-CDs. X-ray photoelectron spectroscopy (XPS) and Fourier. Transformed infrared (FTIR) absorption were measured to investigate the composition and chemical structure of the SL-CDs. As seen in the FTIR spectra (Fig. 3a), the peak centered at 3284 cm−1 is arising from the stretching vibration of –OH and H2N- groups. The stretching vibration of CeH bonds give rise to the appearance of the band at 2930 cm−1. The peak located at 1596 cm−1 originates from the bending vibrations of NeH. The 1401 cm−1 peak is contributed by the CeN stretching vibrations. The peak situated at 1245 cm−1 is identified to be the CeS stretching vibrations. The CeO stretching vibrations generate the peak at 1026 cm−1. The XPS spectrums provide rich information about the surface functionalization stage of SL-CDs. The XPS survey spectrum of the SL-CDs exhibited four prominent peaks originating from C1s, O1s, N1s, and S2P (Fig. 3b). The percentage of C, O, N, and S is 57.63%, 37.41%, 3.98% and 0.98%, respectively. The C1s XPS spectrum revealed that there are four different types of carbon atoms (Fig. 3c), that is, C=C/C-C with a binding energy at 284.5 eV, CeO at 286.4 eV, C]O at 287.9 eV, and C-N/C-S at 285.2 eV. The N1 s XPS spectrum showed two peaks at 400.1 eV and 401.1 eV (Fig. 3d), which
The prominent absorption peak located at 280 nm in the UV–Vis absorption spectrum is ascribed to the π-π* transition of C]C [36]. The PL spectrum of SL-CDs showed a strong peak at 455 nm when the excitation wavelength is 390 nm. The diluted solution of the SL-CDs is light yellow colour under sun light, while turns to blue under 365 nm ultraviolet lamp illumination (Fig. 4a). The PL spectrum of the SL-CDs showed obvious excitation wavelength-dependent property, as usually observed by previous publications for fluorescent carbon materials. This excitation wavelength-dependent property is caused by the fact that the surface state of the SL-CDs can affect the band gap of SL-CDs [37]. The surface state of the SL-CDs is analogous to a. Molecular state; while the size effect is induced by the quantum confinement effect when the size of the SL-CDs is pretty small. The surface state and the size effect coordinate together to endow the complex PL properties of the SL-CDs [38]. The PL emission peaks are red-shifted from 425 nm to 525 nm as the excitation wavelength changed from 340 nm to 470 nm; The PL intensity also changed (Fig. 4b). These PL properties of the SL-CDs make the CDs suitable to be used in optical bioimaging in vitro and in vivo, especially after considering the formative needs for molecular probes in cellular imaging [39]. The PL QY of SL-CDs was calculated to be approximately 18.6% at the excitation wavelength of 360 nm by using the PL QY of quinine sulfate as reference. The measured absorbance of SL-CDs and quinine sulfate at 360 nm for QY calculation were 0.026 and 0.011, respectively. The corresponding integrated fluorescent emission intensities were 4095 and 5030, respectively. The corresponding fluorescence emission spectra under the excitation wavelength of 360 nm are depicted in Fig.S2. This QY value of the SL-CDs prepared in our work is
Fig. 2. a Transmission electron microscopy images of SL-CDs, b Particle diameter distribution of SL-CDs. 146
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Fig. 3. a The FT-IR spectra of the SL-CDs, The XPS (b), C1s (c), N1 s (d), O1s (e) and S2p (f) spectra of the as-attained SL-CDs.
Fig. 4. a UV–Vis absorption and PL emission spectra of the aqueous solution of the SL-CDs (Insets show the photographs of SL-CDs under visible light (left) and UV light (right), b PL spectra of the SL-CDs at varied excitation wavelength ranging from 340 to 470 nm.
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ions over other competitive cations including Zn2+ ions. To evaluate the detection limit for Cd2+ detection, the medium conditions were further optimized. The influence of pH value of the medium on the detection sensitivity of Cd2+ ions was investigated (Fig. S6a). We found that the optimum quenching efficiency was achieved when the pH value of the solution is around 5.0. To find the best buffer solution, the effects of four different buffer solutions with a pH value of 5.0 were further studied (Fig. S6b). Among them, HAc-NaAc (ACE) buffer solution was found to be the best buffer medium as the highest △F is obtained when ACE is used. Therefore, the ACE buffer solution with a pH value of 5.0 was used in this work. The ratio of F/F0 keeps constant when the reaction time is ≥180 s (Fig. S6c), demonstrating that the detection of Cd2+ ions using SL-CDs only needs a short time. 180 s was chosen as the reaction time for further studies. As expected, the PL intensity is reduced gradually as the concentration of Cd2+ ions increases (Fig. 6a), implying that the PL intensity of the SL-CDs is very sensitive to Cd2+ ions. The relationship between the concentration of Cd2+ ions and the ratio of F/F0 follows two lines in different concentration ranges of Cd2+ ions (Fig. 6c and d). When the concentration of Cd2+ ions is in the range of 0.1–3.0 μM, the relationship can be described by F/F0 = 1.00084–0.15702 × [Cd2+] (R2 = 0.98586), and the corresponding binding constant is 2.816 × 105 ± 11,670 L mol−1. When the concentration is in the range of 5.0–30.0 μM, the relationship is F/F0 = 0.51008–0.00588 [Cd2+] (R2 = 0.99397), and the corresponding binding constant is 3.636 × 104 ± 2120 L mol−1. The detection limit of Cd2+ ions can reach as low as 15 nM at a signal-to-noise ratio (S/N) of 3. The obtained detection limit is comparable to previously reported sensing methods in the literature (Table S2). To investigate the potential of our detection method in real cases, we studied analysis of the tap water samples. As shown in Table 1, it can be seen that the results of recovery for the samples are satisfied.
higher than those of CDs reported previously (Table S1), which demonstrates the extraordinary fluorescent properties of the SL-CDs [40]. The high PL quantum yield of the SL-CDs is beneficial to the applications in cell imaging and detection Cd2+ ions as shown in the following content. The PL properties of the SL-CDs under different pH values were further studied (Fig. S3a). The PL intensity of the SL-CDs increases as the pH value was increased from 2 to 3, while kept stable as the pH value was further increased from 3 to 9. When the pH was larger than 9, the PL intensity gradually reduced. This means that the SL-CDs can be used as a pH sensor to monitor the pH distribution within cells. The effects of pH on the absorption spectral changes are shown in Fig.S3b. The absorption peak showed no obvious change. The absorbance value of the SL-CDs increases as the pH value was increased from 2 to 3, while kept stable as the pH value was further increased from 3 to 9. When the pH was larger than 9, the absorbance value gradually reduced. The PL intensity did not show significant change when the solution containing different amounts of NaCl (Fig. S3c). More importantly, the PL intensity of the SL-CDs did not reduce when continuously irradiated under a 365 nm UV-lamp with the distance of 15 cm between the sample and light source (Fig. S3d), demonstrating the fascinating photostability of the fluorescence probe. The photostability of the SL-CDs makes them promising for application in bioimaging.
3.3. SL-CDs Nanoprobe for Cd2+ Detection To thoroughly evaluate the selectivity of the SL-CDs in detecting different metal ions, the PL intensity variations before and after introducing 50 μM of metal ions, including Cs+, Cd2+, Hg2+, Cu2+, Pb2+, Al3+, Fe2+, Zn2+, Ba2+, Mg2+, Ca2+, Co2+, and Fe3+ into the SL-CDs solution were investigated separately. As shown in Fig. 5a and Fig.S4, the PL of the SL-CDs is dramatically quenched after. The addition of Cd2+ ions. In a sharp contrast, no or very unobvious PL quenching phenomenon is observed when other metal ions are introduced into the colloidal solution of SL-CDs. This means that the SLCDs can be used as an efficient fluorescence probe for Cd2+ detection. The obvious fluorescence quenching of the SL-CDs after Cd2+ addition may be owing to the special binding between the Cd2+ ions and the phenolic hydroxyl and/or amine groups of SL-CDs. Moreover, we measured the fluorescence intensity of SL-CDs in the presence of CdCl2, Cd(NO3)2, and Cd(SO4)2 at the same concentration to eliminate the effects of Cd2+ counter ions (Fig.S5). The obtained results indicate almost the same PL quenching phenomenon. Thus, the changes were not due to the specific counter ion of Cd2+. The interference test results were shown in Fig. 5b, demonstrating that the other interference metal ions showed no obvious effect on the PL intensities of the SL-CDs. Nevertheless, once Cd2+ ions are introduced into the testing solutions, significant fluorescence quenching is observed. These results confirm that the SL-CDs have an excellent selectivity during detection of Cd2+
3.4. Possible Mechanism of the Fluorescence Response of SL-CDs to Cd2+ To deepen our understanding about the PL quenching mechanism of. SL-CDs by Cd2+, UV–Vis absorption spectra, the effects of temperatures on quenching reaction, the lifetime of the PL and the quenching rate constant Kq were recorded. As shown in Fig.7a, the absorption peak of pure SL-CDs is centered at 280 nm. Nevertheless, this absorption peak gets weakened significantly when Cd2+ was introduced into the colloidal solution of SL-CDs, whereas no obvious absorption spectral changes were observed with the addition of other metal cations (Fig.S7). We conclude that this process is a static quenching process, because dynamic quenching only affects the excited state of fluorescent molecules and has no effect on ultraviolet absorption [41]. And the hydroxyl at surface of SL-CDs can react with metal ions, thus can infer a non-luminous ground state compounds were Fig. 5. a Fluorescent response of SL-CDs to different metal ions in HAc-NaAc buffer solution (pH 5.0). 50 μmol/L is used for each metal ion. F0 and F represent the fluorescent intensity of SL-CDs at λem of 455 nm excited at 390 nm in the inexistence and existence of metal ions, respectively, b The fluorescent intensity of SL-CDs (red) and SL-CDs + Cd2+ (blue) solutions in the existence of various metal ions (50 μmol/L). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. a PL emission spectra of SL-CDs upon addition of various concentrations of Cd2+ in HAc-NaAc (pH = 5) at an excitation wavelength of 390 nm. Inset: Plot of the fluorescent intensity against Cd(II) concentration within the range of 0–50 μM, b Sensing principle of the SL-CDs based probe for Cd2+, The linear relationship of F/F0 against the Cd2+ concentration over the range of (c: 0.1–3.0 μM; d: 5.0–30.0 μM).
molecular motion accelerates. The result will increase the diffusion coefficient of the molecule. Finally, the degree of quenching is increased. On the contrary, the stability of the complex may decrease as the temperature is increased, so the degree of static quenching is reduced. According to these, the quenching of Cd2+ on SL-CDs should be static quenching, this is consistent with the results of ultraviolet absorption. The interaction of quenching agent and the excited state of fluorescence substance leads to dynamic quenching, and the interaction with the ground state of fluorescence substance leads to static quenching [41]. The dynamic quenching process is the process of competing with spontaneous emission process to shorten the lifetime of excited molecules. For static quenching, the existence of quenching agent will not change the lifetime of excited fluorescence molecules. The fluorescent lifetime of SL-CDs is determined to be 3.43 ns, which turns to be 3.41 ns after the addition of Cd2+ ions (Fig.7b). The slightly reduced fluorescent lifetime demonstrates that the mechanism of Cd2+ quenching of SL-CDs is a static quenching. So, compared with other metal ions, Cd2+ ions have more cogent affinity and faster chelating kinetics towards the hydroxyl on the surface of the SL-CDs.
Table 1 Detection of Cd2+ in Tap water. Sample
Added (μmol/L)
Found (μmol/L)
Recovery (%, n = 5)
Tap water
0.2 0.5 2.5 10 20
0.188 ± 0.008 0.489 ± 0.007 2.575 ± 0.009 9.9 ± 0.008 21.2 ± 0.009
94% 97.8% 103% 99% 106%
formed by Cd2+ with hydroxyl on the surface of SL-CDs. So, the ultraviolet absorption and fluorescence intensity of SL-CDs were reduced. As shown in Fig.S8, the degree of quenching decreases as the temperature was increased from 15 °C to 50 °C. However, the quenching phenomenon is not obvious when the concentration of Cd2+ is low, indicating that Cd2+ and SL-CDs formed a nonluminous complex. More and more nonluminous complex are formed as the Cd2+ concentration was increased. So, the quenching becomes more and more obvious. The dynamic quenching is related to diffusion coefficient. The viscosity of the solution decreased when the temperature was increased, and the
Fig. 7. a UV–Vis absorption spectra of the Cd2+ ions (black line), SL-CDs (blue line) and SL-CDs after exposing to Cd2+ ions (red line). b Fluorescent decay traces of the SL-CDs in the inexistence (black line) and existence (red line) of Cd2+ ions, (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 8. Confocal fluorescent pictures of A549 cells incubated with SL-CDs for 24 h under (a) 405 nm excitation and (b) bright field, (c) The merged image of a and b.
In order to further study the quenching process between Cu2+ and carbon dots, The quenching rate constant Kq is calculated by Kq = Ksv/ τ0, wherein, Ksv is Stern-Volmer quenching constant, which is the binding constant of the complex formed by quencher and fluorescent substance for static quenching. τ0 is the fluorescence lifetime of carbon dots without quenching agent. The calculated value of Kq is 8.2 × 1013 L/(mol·s), which is 1000 times higher than the upper limit of the typical dynamic quenching rate constant 1.0 × 1010 L/(mol·s), indicating that the quenching of carbon dots by Cd2+ is caused by the formation of the non-luminescent complex, which is static quenching process.
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3.5. Cytotoxicity and Cellular Imaging The cytotoxicity of luminescent quantum dots is important for living cell imaging applications. Therefore, to explore the potential application of SL-CDs in living cell imaging, the cytotoxicity of SL-CDs is first evaluated by the MTT assay in A549 cells. The A549 cell viabilities were evaluated after the cells are exposed to SL-CDs at different concentrations. The SL-CDs have negligible or extremely low cytotoxicity (Fig. S9). Almost 100% of the cells retain viable even after exposing SLCDs at a concentration of up to 10 μg/mL. There are still 92% of the cells viable after exposing SL-CDs at a concentration as high as 50 μg/ mL. Therefore, the SL-CDs are safe for in vitro and in vivo cell imaging applications. Moreover, a prominent intracellular fluorescence is observed after the SL-CDs incubated with A549 cells. Fig. 8 displayed the samples observed under bright field and excited at 405 nm by a fluorescence microscopy. The result demonstrated that the fluorescent signals are from the perinuclear regions of the cytosol, indicating the outstanding cell-permeability of the SL-CDs into living cells owing to their beneficial surface functional groups and small size. 4. Conclusion In summary, a facile method was developed to synthesize N- and S co-doped carbon dots using scallion as the carbon source. The obtained SL-CDs exhibit desired fluorescent properties with a PL QY of 18.6%, which makes them be able to serve as an effective fluorescence probe for sensitive detection of Cd2+ ions. The detection limit can reach as low as 15 nM. Detection of Cd2+ ions in environmental water demonstrated this sensor can be successfully used to monitor the concentration of Cd2+ ions in daily used water. Eventually, the low toxicity and strongly fluorescent carbon dots were applied for cell imaging and the quenched fluorescence by adding Cd2+, demonstrating their potential towards diverse applications. Acknowledgements The authors would like to thank a Grant from the Natural Science Foundation of Jiangsu Province (BK20170175). 150
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