Author’s Accepted Manuscript Synthesis of highly photoluminescent carbon Dots via citric acid and Tris for Iron (III) Ions Sensors and Bioimaging Zhulong. Zhou, Ming. Zhou, Aihua Gong, Yan Zhang, Qijun Li www.elsevier.com/locate/talanta
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S0039-9140(15)00253-2 http://dx.doi.org/10.1016/j.talanta.2015.04.015 TAL15515
To appear in: Talanta Received date: 10 February 2015 Revised date: 27 March 2015 Accepted date: 5 April 2015 Cite this article as: Zhulong. Zhou, Ming. Zhou, Aihua Gong, Yan Zhang and Qijun Li, Synthesis of highly photoluminescent carbon Dots via citric acid and Tris for Iron (III) Ions Sensors and Bioimaging, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis of Highly Photoluminescent Carbon Dots via Citric Acid and Tris for Iron (III) Ions Sensors and Bioimaging Zhulong Zhou1, Ming Zhou2*, Aihua Gong1, Yan Zhang2, Qijun Li2 1
Center for Photon Manufacturing Science and Technology, Jiangsu University, Zhenjiang 212013, China. The State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 10084, China.
*Corresponding author. Tel: +86 010 6278 3968. E-mail addresses: [email protected]
Abstract In this work, high quantum yield and strong photoluminescent carbon quantum dots (C-QDs) are successfully synthesized via a facile and green hydrothermal method using Citric acid and Tris as precursors. The as-synthesized C-QDs with a quantum yield (QY) as high as 52% were characterized by UV, FT-IR, TEM, XPS and fluorescence spectroscope. TEM results show that C-QDs are mono-dispersed spherical particles and the diameter distribution of C-QDs is 2.8±1.1 nm. The extraordinary photoluminescent properties and low cytotoxicity of C-QDs were obtained through optical property characterization and cytotoxicity assay. In addition, we found that the as-prepared C-QDs had a high affinity for Fe3+ ions and the response toward Fe3+ ions was highly linear (R2=0.997) over the concentration range from 2 to 50 μM, which could provide an effective platform for portable detection of Fe3+ ions. Also, it is demonstrated that the photoluminescent C-QDs display hypotoxicity and are biocompatible for use as biosensors in living cells.
Key words: Carbon quantum dots; Hydrothermal; High quantum yield; Fe3+ ions detection; Bioimaging 1. Introduction Recently, photoluminescent carbon-based materials, also known as carbon quantum dots (C-QDs), have received tremendous attention due to exceptional advantages such as low toxicity [1-2], high chemical stability , excellent biocompatibility [4-6], high-fluorescence . Considering these unique physical-chemical characteristics, C-QDs have been applied widely in fields of catalysis , printing ink , biological sensors [10-12], bioimaging  and drug delivery . Since 2006, Sun et al. found a new fluorescent nanoparticle named as carbon dots, many approaches have been found to prepare C-QDs . To date, a series of methods for obtaining carbon-based materials have been developed, such as chemical oxidation method , ultrasonic method , hydrothermal synthesis [6 18-20], solvothermal method , microwave method  and laser ablation method . Du et al. 1
provided the 19.2% QY of C-QDs obtained by the hydrothermal treatment of citric acid and 2-(2-aminoethoxy)-ethanol . Zeng et al. reported that fluorescence C-QDs with 43.8% QY for sensitive and selective detection of iodide . However, it is still a problem for progress in controlling the morphology, particle size, and surface chemistry of the resultant products with high quantum yields. Efficient one-step strategies for the fabrication of C-QDs on a large scale are still a challenge in this field. In addition, the mechanism of C-QDs associated photoluminescence is not clear completely. So far, the speculation reason of the luminescence might be the radiative recombination of the energy-trapping sites on the C-QDs surface . To our knowledge, Fe3+ ions are very essential because it plays an important role in clinical and environmental . The presence of Fe3+ ions in biological systems has to be efficiently moderated and monitored . Many of papers have reported fluorescent probes for Fe3+ ions based on fluorescence quenching response. For example, Qu and co-workers recently reported the fluorescence of C-QDs can be quenched by Fe3+ ions with a good linear correlation (R2=0.98) and the detection limit of 0.32 μM . Lu et al. performed a fluorescence emission titration experiment, the fluorescence of C-QDs almost complete quenching with the gradual addition of Fe3+ ions . However, the mechanism research of interaction between C-QDs and Fe3+ ions are not thoroughly investigated. We need to further explore the mechanism and the limit of detection for different C-QDs with Fe3+ ions. Herein, a facile and highly photoluminescent strategy is demonstrated for hydrothermal synthesis of C-QDs that by using citric acid and Tris as precursor for the first time (Figure 1). This method is highly reproducible and the sources of the C-QDs have the advantage of being relatively cheap and environmental friendly. Various parameters related to the synthetic process have been investigated and optimised to obtain the best optical property of the C-QDs (Figure S1). The prepared C-QDs, exhibit strong fluorescence within ultrapure water, in the presence of Fe3+ ions can bind with the C-QDs and quench the fluorescence of C-QDs with high sensitivity. Additionally, the fluorescence C-QDs exhibit hypotoxicity and are biocompatible for use as biosensors in living cells. Fig. 1.
2. Experimental Section 2.1 Materials. Citric acid, Tris(hydroxymethyl)methyl aminomethane (Tris), NaOH, KCl, NaCl, CaCl2, CuCl2, MnCl2, MgCl2, NH4Cl, FeCl3，ZnCl2, NiCl2, and FeCl2 were purchased from Beijing Chemical Works. Quinine sulfate was purchased from Sinopharm Chemical Reagent Co., Ltd. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was acquired from Sigma-Aldrich. Human MCF-7 breast cancer cells (MCF-7) was obtained from 2
the American Type Culture Collection (ATCC, Manassas, VA). All other reagents and chemicals were of analytical grade and used without further purification. Ultrapure water with the resistivity of greater than 18 MΩ-cm was used in the following experiments. 2.2 Preparation of C-QDs. C-QDs were prepared by hydrothermal synthesis. First, 0.21 g citric acid and 0.12 g Tris were dissolved in 10 mL ultrapure water thoroughly to form a clear solution. Then, the solution was transferred into a poly(tetrafluoroethylene) (Teflon)-lined autoclave (30 mL) and heated at a specific temperature (250℃) for 6 hours. After the reaction, the reactors were cooled down at room temperature. The deep-brown aqueous dispersion formed was centrifuged at high speed (12000 rpm/min) for 15 min in order to remove any insoluble particulates. Then the upper brown solution was dialyzed against ultrapure water through a dialysis membrane (MWCO=1000 Da) to remove the precursors that did not participate in the reaction and resulting small molecules. At last, the product in the dialysis membrane was dried by a freezing dryer. The drying product was dissolved into a certain amount of solution (1mg/mL) and stored in the refrigerator for further research. 2.3 Quantum yield measurements. The quantum yield (QY) of C-QDs was measured according to the method. Briefly, quinine sulfate (0.1 M H2SO4 as solvent), which QY is about 54.2%, was chosen as a reference standard. The absorbance for the quinine sulfate and the C-QDs at the 330 nm excitation and the fluorescence spectra of the same solutions at the same excitation were measured respectively. Then the integrated fluorescence intensity from the fluorescence spectrum was calculated. At last, the QY of C-QDs was calculated according to a comparative equation : Y𝑢 = 𝑌𝑠 ∙ (𝐹𝑢 ⁄𝐹𝑠 ) ∙ (𝐴𝑠 ⁄𝐴𝑢 ) ∙ (𝜂𝑢2 ⁄𝜂𝑠2 ) Where Y is the QY, F is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the absorbance. The subscript "s" and "u" refers to standard and the sample. For these aqueous solutions, ηu /ηs=1. It should be noted that the excitation wavelength for measurements of standard was set at the same relevant wavelength of the C-QDs. 2.4 Apparatus. Transmission electron microscope (TEM) image of C-QDs was obtained with H-7650B electron microscope (Hitachi, Japan). The fluorescence and the absorption spectra were recorded with F-7000 (Hitachi, Japan) fluorescence spectrophotometer and UV-3900 (Hitachi, Japan) spectrophotometer, respectively. The Fourier transform infrared spectroscopy (FT-IR) spectra of C-QDs were recorded on a Nicolet 6700 FT-IR spectrometer (Hitachi, Japan) with a crystal, over the range of 1000–4000 cm-1. Raman spectra were measured with a Horiba 3
Raman system model HR800 spectrometer with radiation at 532 nm. X-ray Photoelectron Spectroscopy (XPS) was investigated by using PHI-5300 X-ray Photoelectron spectrometer with a mono X-ray source Mg Kα excitation (1253.8 eV). The cytotoxicity of C-QDs was recorded by a microplate reader (Thermo Scientific Varioskan Flash, USA). The confocal fluorescence images of C-QDs labeled cells were acquired with a fluorescence confocal microscope (Zeiss LSM 780, Germany) under ambient conditions. 2.5 PH effect on fluorescence emission of C-QDs. A dilute solution of C-QDs (0.1mg/mL) in ultrapure water was prepared by using ultra-sonication. 100 μL C-QDs solution was diluted to 3 mL by using different solutions with pH 1.5–13 subsequently. The pH values of these solutions were tuned by HCl (10 mM) or NaOH (10 mM) solution and final measured by pH meter. Then the fluorescence spectra of these solutions were recorded by using a F-7000 spectrometer. 2.6 Fe3+ ions detection. The detection of Fe3+ ions were conducted in phosphate buffer saline (PBS, 10 mM, pH=7) solution. Meanwhile, some representative cations, including K+, Na+, Ca2+, Cu2+, Mn2+, Mg2+, NH4+, Fe3+, Zn2+, Ni2+, and Fe2+ were chosen to detect the fluorescence intensity of C-QDs. What’s more, the reagents are all contain Cl- ions, so it can avoid the influence of other difference anions. The above mentioned cations solution (10 mM) were prepared with PBS solution. Then, 50 μL C-QDs was added into 3 mL cations solution to form homogeneous solution. Additionally, Fe3+ ions was diluted various multiples to verify the selectivity of C-QDs. The fluorescence spectra were measured after reaction for 10 min. All experiments were performed at room temperature. 2.7 Cytotoxicity and Cell imaging. The cytotoxicity of C-QDs was evaluated by MTT assay. In the experimental process, MCF-7 cells (1×104 cells/mL) were seeded in 96-well plates and incubated for 24 h at 37 °C under 5% CO2. Then a series of concentrations of C-QDs were added into wells with a range from 0 mg/mL to 1 mg/mL. After incubation for 24 h, 20 μL of 5 mg/mL of MTT solution was added into each well and incubated for another 4 h. After replacing the supernatant with 150 μL DMSO, the absorbance of each well was recorded at 490 nm by a microplate reader. In order to obtain actual cell imaging, MCF-7 cells were seeded in 24-well plates at a density of 1×104 cells/mL and incubated at 37 °C for 24 h. Then 20 μL of 0.5 mg/mL of C-QDs solution was added into each well and incubated for another 24 h. After incubation, the cells were washed thrice with PBS, followed by fixed with 40% of phosphate-buffered paraformaldehyde for 10 min. Finally, the cells were washed twice with PBS solution and images were captured with Zeiss LSM 780 confocal lasers scanning ﬂuorescence microscopy.
3. Results and Discussion 4
3.1 Characterization of the C-QDs. The morphology and structure of C-QDs were characterized by a series of instruments. Transmission electron microscopy (TEM) reveals nearly spherical nanoparticles of C-QDs 2−4 nm in size (Figure 2a). The Figure 2b histogram shows the diameter distribution of C-QDs is 2.8±1.1 nm. In the UV-Vis spectra (Figure 2c), weak absorption peaks were observed at 238 nm and 330nm in an aqueous solution of C-QDs. These absorption shoulders may be attributed to the π–π* transition of the C=C bonds, the n–π* transition of C=O bonds or others. The bright blue luminescence and highly transparent of C-QDs solution can be clearly observed under UV irradiation and ambient light, respectively (Figure 2c, inset). The fluorescence emission spectra of C-QDs were primarily investigated under excitation wavelengths. From the fluorescence spectra (Figure 2d), C-QDs have wonderful emission under excitation wavelength from 300 nm to 400 nm that was similar to previous report . This phenomenon is common and contributed to the surface state affecting the band gap of C-QDs. The surface state is analogous to a molecular state whereas the size effect is a result of quantum dimensions, both of which contribute to the complexity of the excited states of C-QDs . Particularly, the 52% of QY of the C-QDs was calculated at 330 nm optimal excitation according to the above comparative equation. This result is higher when compared with previous report [23-24]. Fig. 2. According to Raman spectra of C-QDs, No obvious D-band (~1360cm-1) or G-band (~1580cm-1) was observed with such a low carbon-lattice-structure (Figure S2). The chemical bonds and functional groups on the surface of C-QDs were obtained from the FT-IR spectra (Figure 3a). In the FT-IR analysis, the absorption bands at 3390 cm-1 and 3250 cm-1 corresponding to stretching vibration of O-H and N-H, respectively. The 2930cm-1 corresponding to stretching vibration of C-H, the 1700cm-1 and 1020cm-1 are attributed to C=O and C-N stretching vibration. The 1560cm-1 could be attributed to bending vibration of N-H. Additionally, the image reveals the characteristic absorption bands of C-NH-C at 1400 cm-1. It is demonstrated that carboxylic groups on the surface of C-QDs have been converted into amide groups during the reaction process. The above analysis shows that C-QDs contain -OH, -COOH, and -NH2 groups. Meanwhile, the zeta potential of C-QDs was -6.5 mV, suggesting that C-QDs are negatively charged due to carboxylic groups, which also support this result indirectly. Fig. 3. More information on differences in surface functional groups of the C-QDs is further provided by XPS analysis (Figure 3b). XPS survey spectra show that Carbon (C 1s, 284.8 eV), Nitrogen (N 1s, 400.6 eV) and Oxygen (O 1s, 532 eV) elements of C-QDs. The partial XPS spectrum of C 1s (Figure 3c) can be divided into four 5
component peaks, which shows the presence of C-C (284.7 eV), C-O (286 eV), C=O (287.4 eV) and C-N (285.6 eV). According to the C 1s spectra, the relative contents of the above chemical states were calculated by their integral. The contents of C-C, C-O, C=O and C-N are 59.3%, 26.6%, 9.5% and 4.6%, respectively (Table S1). Moreover, as shown in the O 1s spectrum (Figure 3d), two peaks were observed at 531.0 eV and 532.6 eV, which were attributed to C-O and C=O, respectively. 3.2 PH effect on fluorescence emission of C-QDs. The luminescence of C-QDs is also investigated under different pH range from 1.5 to 13 solutions. Figure 4 shows that the fluorescence intensity of C-QDs measured with excitation wavelength of 330 nm is pH dependent. Different pH values had distinct effects for the fluorescence intensity of C-QDs. Under strong acidic or alkaline condition, the photoluminescence were greatly weak. The fluorescence intensity of C-QDs first increases with a rise of pH from 1.5 to 7 and then decreases with a further increase. The highest fluorescence intensity is observed at pH=7 solution. In order to obtain a better detection for Fe3+ ions, the buffer of PBS (10 mM, pH=7) was used for the Fe3+ ions detection. According to this phenomenon, the C-QDs was used as fluorescent ink at pH=7(Figure S3). Fig. 4. 3.3 C-QDs nanoprobe for Fe3+ ions detection. Qualitative and quantitative detection of Fe3+ ions are very essential because it plays an important role in clinical and environmental. As show in Figure 5a, under the presence and absence of representative cations, the influence of cations on the fluorescence spectra of C-QDs was evaluated by recording the fluorescence intensities. The blank position of the emission was not affected upon addition of cations. Obviously, there was a seriously decrease in fluorescence intensity when the solution contains Fe3+ ions. For Cu2+, Ni2+ and Fe2+ ions, a minor fluorescence decrease was observed. These phenomena maybe were caused by a metal-catechol interaction  or metal-quenching effect . These results confirm that the C-QDs show an excellent selectivity toward Fe3+ ions over other competitive cations, which is owing to the binding of C-QDs with Fe3+ ions. Fig. 5. For the further sensitivity research, the dependence of the quenching effect (F0/F) was investigated under the different concentrations of quenching Fe3+ ions. Figure 5b reveals the quenching effect of the Fe3+ ions on the fluorescence of the C-QDs at 330 nm excitation wavelength. The fluorescence intensity of C-QDs decreased upon increasing the concentration of Fe3+ ions, revealing that this system is sensitive with Fe3+ ions. As previously reported, strong quenching of fluorescence by Fe3+ ions might be due to the effective coordination or chelation 6
interactions between Fe3+ ions and the plentiful -OH, -NH2, and -COOH groups on the surface of C-QDs [6 31]. This phenomenon may lead to the formation of nonradiative electron-transfer or energy-transfer and resulting in the substantial fluorescence quenching. More importantly, the fluorescence quenching data follows the Stern–Volmer equation : 𝐹0 ⁄𝐹 − 1 = 𝐾𝑠𝑣 𝐶 Where F0 and F are the fluorescence intensities of the C-QDs in the absence and presence of Fe3+ ions solution, respectively. Ksv is the Stern–Volmer quenching constant and c is the analyte (Fe3+ ions) concentration. A good linear correlation (R2=0.997) is observed over the concentration range of 0–50 μM. Meanwhile, the detection limit for C-QDs is 1.3×10−6M at a signal-to-noise ratio of 3. It indicates that the C-QDs probes had a high affinity for Fe3+ ions and the response toward Fe3+ was highly linear over the concentration range from 2 to 50 μM. Besides, the fitted linear data (Figure 5c) could be expressed (Table S2). To evaluate the potentials of this probe system’s applicability in real samples, we applied the equal method to the analysis of the aqueous samples (tap water and lake water). As show in Table S3, it can be seen that the results of recovery for the samples are satisfied. 3.4 Cytotoxicity and Cellular imaging. An ideal fluorescent probes for applications should be high fluorescent, biocompatible, non-toxic to biological systems, and be stable against photobleaching. For further research on biological applications of C-QDs, The cell viabilities of Human MCF-7 breast cancer cells (MCF-7) cells were tested after being exposed to C-QDs at a series of concentrations. Figure 6a shows that the cell viabilities of MCF-7 cells declined to 80 % upon addition of the C-QDs at up to 1 mg/mL, which suggests these C-QDs have extremely low cytotoxicity and biocompatibility. Moreover, a remarkable intracellular fluorescence is obtained after the C-QDs are incubated with MCF-7 cells. Figure 6b, c, d and e exhibit the samples were observed under bright field (b) and excited at 405 nm (c), 488 nm (d) and 514 nm (e) by a laser scanning confocal microscope. The result reveals that the fluorescence signals are from the perinuclear regions of the cytosol, indicating excellent cell-permeability of the C-QDs into living cells. Fig. 6.
4. Conclusion In this study, a facile and highly photoluminescent strategy is demonstrated for hydrothermal synthesis of C-QDs by using citric acid and Tris as carbon sources. The obtained C-QDs with a quantum yield as high as 52% show high water-soluble, good mono-dispersed stability and strong blue photoluminescent. The research found that the fluorescence intensity of C-QDs is pH dependent with a rise of pH from 1.5 to 7 and then decreases with a 7
further increase. In addition, a simple, reliable and sensitive Fe 3+ ions detection was rendered by the C-QDs sensing system. Our C-QDs probes provided highly sensitive analyses of Fe3+ ions with a detection limit of 1.3 μM and a good linear correlation (R2=0.997) over the concentration range of 0–50 μM. In addition, we also obtained the concentration of Fe3+ ions in the tap water and lake water by using these probes. Besides, the C-QDs also make it has potential application value in the field of biological imaging and other applications fields because of its excellent optical properties.
Acknowledgements This research was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB013004), Major Project of State Key Laboratory of Tribology (Grant No. SKLT2014A01).
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The legends for the figures
Figure 1. Scheme proposed for Synthesis of the water-dispersible C-QDs and application
Figure 2. (a) TEM image of the C-QDs; (b) The corresponding dot size distribution histogram of C-QDs; (c) UV-Vis absorption and the best excitation fluorescence; (d) Fluorescence spectra of the C-QDs under excitation wavelength from 300 nm to 400 nm.
Figure 3. (a) FT-IR spectra of C-QDs (1-citric acid, 2-Tris, 3-C-QDs); (b) XPS spectra of C-QDs; 10
High-resolution XPS spectra of C 1s (c) and O 1s (d).
Figure 4. PH effect on fluorescence emission of C-QDs under 330 nm excitation wavelength. The 10 mM HCl solution was used to tune when pH 7. All the pH values are the average values.
Figure 5. (a) Selectivity of the C-QDs toward Fe3+ ions; All the concentrations of the cations (10 mM) were prepared with PBS (10 mM) buffer at pH=7; excitation wavelength = 330 nm; (b) Representative fluorescence emission spectra of C-QDs in the presence of increasing Fe3+ ions concentrations (0–200 μM) in PBS buffer (10 mM) at pH 7; (c) The relationship between F0/F-1 and Fe3+ from 0 to 20 μM; (d) The relationship between F0/F-1 and Fe3+ ions from 0 to 200 μM. F and F0 are the fluorescence 11
intensities of C-QDs at 330 nm in the presence and absence of Fe3+ ions, respectively
. Figure 6. (a) Cytotoxicity of C-QDs. Fluorescence microscopy images of MCF-7 cells labeled with the C-QDs: (b) excitation by bright field; (c) excitation by 405 nm; (d) excitation by 488 nm; (e) excitation by 514 nm.
Highly fluorescent quantum yield carbon quantum dots (C-QDs) were prepared.
The C-QDs fluorescence was selectively quenched by Fe3+ ions.
The C-QDs acted as sensitive and selective nanoprobes for Fe3+ ions determination.
The C-QDs were applied to optical bioimaging of MCF-7 cells with low cytotoxicity.