Highly Photoluminescent Nitrogen-Doped Carbon Nanodots and Their

Dec 7, 2015 - Finally, the absorption at 490 nm was measured by Elx800 Micropalte reader (BioTek, USA). 2.5Protective Effect. Cells were cultured in 9...
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Highly Photoluminescent Nitrogen-Doped Carbon Nanodots and Their Protective Effects against Oxidative Stress on Cells Zi-Qiang Xu, Jia-Yi Lan, Jian-Cheng Jin, Ping Dong, Feng-Lei Jiang, and Yi Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08945 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 8, 2015

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Highly Photoluminescent Nitrogen-Doped Carbon Nanodots and Their Protective Effects against Oxidative Stress on Cells Zi-Qiang Xu#1,2, Jia-Yi Lan#1,Jian-Cheng Jin1,Ping Dong1, Feng-Lei Jiang1, Yi Liu*,1 1

State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for

Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China 2

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials,

Ministry-of-Education Key Laboratory for the Green Preparation andApplication of Functional Materials, Hubei Province Key Laboratory of Industrial Biotechnology, Faculty of Materials Science & Engineering, HubeiUniversity, Wuhan, 430062, P. R. China.

Key Words: Carbon nanodots, Highly photoluminescent, Nitrogen doped, Protective effects, Oxidative stress *Corresponding author: Tel: +86-27-68753465(O), 68756667(L); Fax: +86-27-6854067 E-mail address: [email protected] (Y. Liu) These two authors contributed equally to this work. 1

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Abstract Highly photoluminescent (PL) (quantum yield=54%) nitrogen doped carbon nanodots (C-dots)have been prepared through one-step carbonizing citric acid and tris(hydroxymethyl)aminomethane and using oleic acid as solvent.The synthesized C-dots are monodisperse with narrow sizedistribution(average 1.7 nm). The PL properties of C-dots are pH dependent and hence using C-dots as sophisticated pH sensor to detect pH values between 7 and 9 can be expected. In addition, the PL intensity of C-dots remains stable under high ionic strength. The C-dots can protect cells from oxidativeinduced stress, which shows potential to expand the biological application of C-dots, especially inmedical treatment.The protective mechanism is associated

with

intracellularreactive

oxygen

species

intracellularsuperoxide dismutase production.

2

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andthe

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1. Introduction Quantum dots (QDs) are nanomaterials with fascinating optical and electronic properties that possess applications in various fields, such as photovoltaic devices, energy-efficient displays, and biomedical markers1. Compared with conventional dyes and fluorescent proteins, QDs are superior in the aspects of bright fluorescence, broad excitation spectrum, highly photostability under physiological environment2. However, most high-performance QDs are made of toxicity elements, such as cadmium (Cd), lead (Pb) and mercury (Hg), which limit their practical application3. Therefore, development of nanoparticles free of toxic heavy element is important and urgent. Carbon nanodots (C-dots) have attracted great attentionsas fluorescent nanoparticles due to their high aqueous solubility, outstanding photoluminescent (PL) properties, favorable biocompatibility, chemical inertness and easy functionalization4-7.These superior properties of C-dots make them potential candidates for various applications, such as bioimaging8-9, medical diagnosis7,

10-12

, catalysis

4, 13-15

, and photovoltaic

devices16.However, many scientific issues need to be addressed before they are applied to practical applications. Up to now, extensive efforts have been devoted to synthesis of C-dots. The synthetic methods can be classified into two types, that are chemical and physical methods17. Chemical methods consist of electrochemical18, thermal treatment 19, hydrothermal or acidic oxidation20, microwave 21, ultrasonic22 and so on. Physical methods include arc discharge 23, laser ablation24, and plasma treatment25. However, current synthetic methods are mainly deficient in accurate controlling of size distribution and surface chemistry, obtaining highly PL materials as well as large scale synthesis. Thus, citric acid (CA) and tris(hydroxymethyl)aminomethane (Tris), served as carbon and nitrogen source, respectively, were used to prepare nitrogen doped C-dots (N C-dots) through a one-step, facile and high output strategy, which is suitable for gram-scale production. And the quantum yield (QY) was as high as ca. 54%, which is higher than most of other works21-23. Moreover, most present works about the biology application of C-dots 3

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mainlyfocuses on the bioimaging 17. So it is important to expand C-dots to novel applications. Meanwhile, they have also found to be both good electron donors and electron acceptors25.Christensen et al. reported that C-dots could act as antioxidants and prooxidants and they have studied the effects of C-dots on generation of singlet oxygen and other reactive oxygen species (ROS) in aqueous solutions in vitro27. It isfound that C-dots can inhibit oxidation of the radical probesunder decomposition of 2,2’-azodiisobutyramidine dihydrochloride(AAPH). However, the antioxidative ability of C-dots has not yet been studied in the cellular level. Meanwhile, regulation of cell oxidant−antioxidant balanced by nanomaterialshas shown great potential for antioxidant therapies28. Therefore, we investigate the potential of applying C-dots to protect cells against oxidative stress. The protective mechanism was also studied in this work. It is of great significance to explore the application of C-dots in biology fields, especially in medical treatment.

2. Experimental Section 2.1 Materials Sucrose,

CA·H2O,

Tris,

oleic

acid,

acetylcholine

chloride

(AchCl),

mercaptosuccinic acid (H2MSA), N-Acetyl-L-cysteine (NAC), ether, ethanol, hydrogen peroxide and potassium chloride (KCl)were purchased from Sinopharm Chemical Reagent Co. (China).3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT)

was

purchasedfrom

Aladdin

reagent

Co.

(China).2,

7-dichlorodihydrofluorescin diacetate (DCFH-DA) was purchased from Sigma co. (USA). Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (China).All reagents were used as received and aqueous solutions were prepared with ultra pure water (18.2 MΩ·cm-1, Millipore). 2.2 Synthesis of C-dots C-dots were prepared as follows: CA·H2O (4g), Tris (3g) and oleic acid (8 mL) were mixed in a three neck flaskequipped with a water separator. The mixture was heated under vigorouslymagnetic stirring. CA·H2O and Tris gradually melted with the increasing of temperature and the color of the solution turned to yellow. In addition, 4

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when temperature reached 170 °C, much water generated and the temperature kept constant at this temperature and unable to rise smoothly. During this period, the water leave the reaction system through water separator. With the water leaving the reaction system, the temperature of reaction system gradually attainedto 215 °C and kept at this temperature for 6 mins. The color of the solution gradually changed to brownish red during this period. After cooling the supernatant, liquid phase was discarded and brownish solid product was obtained at the bottom of flask. The solid product was washed with ethanol for two times followed by extracted with ether several times in order to further remove the remained oleic acid. Finally, the C-dots were purifiedby dialyzing against deionized water using a membrane with1000 MWCO for 12h and then freeze-dried. 2.3 Characterization High resolution transmission electron microscopy (HRTEM)images were recorded on a JEOL JEM-2100 (HR) electron microscopeoperating at 200 kV. X-ray photoelectron spectroscopy (XPS) analysis was carried out on ESCALAB 250Xi. FourierTransform infrared (FTIR) spectroscopy of C-dots were recordedNicolet 360 (USAThermo) FTIR spectrophotometer.Fluorescence analysis was performed on LS-55fluorophotometer (Perkin-Elmer, USA). UNICO 4802 UV-visdouble-beam spectrophotometer was used to measureabsorption spectra of C-dots.Fluorescence decay curves were measured on a FLS92 system (UK, Edinburgh). 2.4 MTT assay The human gastric carcinoma cell (SGC-7901) and human gastricepithelial cell line (GES-1) was cultured in dulbecco’s modifiedeagle medium (DMEM) supplemented with 10% fetal bovineserum, 1% penicillin, and 1% amphotericin at 37 in5% CO2. The cells were seeded into 96-well plate and allowed to adhere for 12 h. Serialdilutions of C-dots with different concentrations were addedinto well. After incubation for 48 h, 200 µLMTT with concentration of 0.5 mg·mL-1 was added into each well, the cells were allowed to grow for 4 h until purple precipitate was visible. The medium was then removed and 150 µL dimethyl sulfoxide was added. The 5

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clusters were vibrated for 10 min to completely dissolve the crystals. Finally, the absorption at 490 nm was measured by Elx800 Micropalte reader (BioTek, USA). 2.5 Protective effect Cells were cultured in 96-well plates for 24 h and then treated asfollows. To evaluate the protective effect of C-dots againstH2O2induced oxidative stress, cells were incubated with C-dots at different concentrations for 24 h and thentreated with 2mM H2O2 for 2 h. After that, the cells were washed with PBS for three times, then the cellular viability was determined by MTT assay mentioned above. 2.6 Generation of ROS detecting The cells were seeded into 6 well-plate and allowed to adhere for 24 h.Serialdilutions of C-dots with certain concentration were addedinto well.After incubation for 24 h, the medium was then removed and the cells were washed with PBS for three times.Then the cell were cultured inserum-free DMEM containing DCFH-DA for 15 mins and the medium was then removed and the cells were washed with PBS for three times. 400 µL trypsin was added to digest for 3 min and then 600 µL DMEM was added. The cells were blew to monodisperse and transferred to 5 mL EP tube. Then they were centrifuged (1100 rpm) for 5 min. The DMEM was removed, 500 µL PBS was added and the cells were analyzed flow cytometry(Accuri C6,BD, USA). 2.7 Detection of intracellular SOD The cells were seeded into 6 well-plate and allowed to adhere for 24 h.Serialdilutions of C-dots with certain concentration were addedinto well.After incubation for 24 h, the medium was then removed and the cells were washed with PBS for three times. 400 µL trypsin was added to digest for 3 min, 600 µL DMEM was added and they were transferred to 5 mL EP tube. Then they were centrifuged (1100 rpm) for 5 min, the medium was removed and 200 µL 10% SDS was added to lysis for 15 min. Then they were centrifuged (13000 rpm) for 15 min and the supernatant was collected. Finally, the intracellular SOD activity was detected by SOD detection kit. 6

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3. Results and Discussions 3.1 Optimization of reaction materials The synthetic method described above can be used to synthesize different C-dots by changing the reaction materials (Table S1).Green emitting or yellow emitting C-dots can also be obtained, by tuning the reaction conditions (365 nm excitation). As shown in Table S1, CA and Tris systemwas found to be the optimal systems for preparing highly PL C-dots, and thus was chosen as reaction materials in the following experiments. To synthesize highly PL C-dots, amine is very important and primary amine is the best dopant to improve the PL QY of C-dots among these three types of amine. However, N-doping usually results in the blue shift of maximum emission wavelength (λem) of C-dots. This result is similar to the conclusion put forward by the Qu et al29. Carboxyl is also very critical to obtain high quality C-dots. If the reactant does not contain any carboxyl, the PL QY of as-prepared C-dots is always less than 12%. In addition, sulfur doping has a slight effect on the PL QY of C-dots. But the influence of sulfur doping onλem is not certain,sulfur doping cause red shift for some C-dots (CD2) while it does not cause any shift in other conditions (CD3、CD4、CD7). This result is not consistent with the conclusion made by Li et al30. They pointed out that the introduced sulfur atoms enhanced the effect of nitrogen atoms on the properties of the doped carbon nanomaterials through a cooperative effect. Then PL QY of C-dots will increase. The PL is the synergistic effect of various factors, such as surface state, surface passivation and carbogenic core. 3.2 Optimization of reaction conditions The reaction conditions including ratio of reactant and reaction time have also been optimized to obtain highly PL C-dots. As shown in Fig. S1a, when the mass ratio of CA·H2O and Tris is between 1:0.5 and 1:2, the optimal ratio is 1:0.75. Although the PL QY of C-dots prepared by ratio of 1:0.5 is close to that of C-dots prepared by ratio of 1:0.75, the former luminescent materials were difficult to be observed by TEM, maydue to the products obtained 7

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under the former conditions are polymer-like dots rather than carbon nanodots. Moreover, as the reaction temperature performed under this ratio could not exceed 200 °C, carbonization reaction was incomplete (section 3.3). In addition, we have found that mass ratio showed no significant influence on theλem. The influence of reaction time on the PL QY of C-dots was studied (Fig .S1b). When the mass ratio was 1:0.75, the PL QY of C-dots increased at first and then decreased with the increasing of carbonization time. The highest PL QY (54 %) was achieved when the carbonization time was 6 min. The decreasing of PL QY may due to certain degrees of aggregation of C-dots that resulted from long carbonization time.In addition, the carbonization time also showed no effect on the PLλem. 3.3 Synthesis of C-dots Nitrogen-doped C-dots of gram scale have been synthesized by carbonization of CA and Tris with oleic acid in one simple step (Fig. 1). A formation mechanism showed as follow:first, at low temperature, polymerization of CA and Tris are took place to form polymer-like dots with high PL QY,mainly attribute to the conjugated fluorophores. Second, as the reaction proceeds to higher temperature, carbonization of polymer-like dots takes place to form carbon nanodots. In our work, when the temperature reached 170 °C, the color of the solution gradually changed to yellow and there were much water generating in the reaction system that came from the dehydration reaction of CA·H2O and polymerization between CA and Tris, so polymer-like dots were formed during this period. While water leaving the reaction system through water separator, the temperature gradually increased and when it reacheed 215 °C, the color of the reaction system gradually turned to brownish red. So carbon nanodots were formed through carbonization of polymer-like dots formed in previous steps during this stage. When the reaction was performed without water separator, the temperature of reaction system remained at 170 °C and was unable to rise smoothly.If the temperature was kept at 170 °Cfor 2h, theluminescent materials were difficult to be observed by TEM even though the PL QY of product was very high (70%). In addition, when the obtained solution was dialyzed for 1day and freeze-dried, there 8

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was few solid product left, so we speculated that only polymer-like dots can be formed and the carbonization of polymer-like dots was unable to proceed under this temperature. This proposed mechanism is similar to that put forwarded by Gianneliset al31. They pointed out a pyrolysis of CA and ethnolamine (EA) took place at180 °Cleading to polymer-like dots with strongly intense PL spectra. At higher temperature (230 °C), a carbogenic core began forming and the PL QY decreasedsignificantly. 3.4 Characterization of C-dots The size distribution and morphology of C-dots were characterized by TEM (Fig. 2a). As shown in Fig. 2a, C-dots are quasispherical andmonodisperse with a narrow size distribution. Accordingly, Fig. S2 shows the size distribution of C-dots. The statistical diameter of C-dots is 1.7±0.6 nm. The size distribution of C-dots agrees well with Gaussian distribution and full width half maximum (FWHM) of the fitted curves are 0.48 nm, which further confirms the narrow size distribution of obtained C-dots. The crystallinenature of both C-dots was investigated by HRTEM. As shown in Fig. 2b, the C-dots do not reveal any clear lattice fringes, indicating amorphous nature of the C-dots32. This was further supported by XRD pattern which expressed a broad peak at around 19 º(Fig. S3). The surface groups of C-dots wereinvestigated by XPSanalysis. The C-dots show three peaks centered at 285.08, 400.08 and 532.08 eV, which can beattributed to C1s, N1s and O1s (Fig. 3a).Element analysis shows that the content of C, N, H in C-dots is

49.63%, 6.43%, 6.38%, respectively. The deconvolution of the C1s spectrum of the CDs indicates the presence of four types of carbon bonds, sp2 C=C (284.7 eV), C-O and C-N (286.2 eV), C=O (287.8 eV) and COO (289.0 eV) (Fig. 3b). Thehigh-resolution spectra of N1s (Fig. 3c) reveal the presenceof both pyridinic type (399.7 eV) and pyrrolic type (400.6 eV)N atoms. We also use FTIR to identify the functional groups of C-dots (Fig. 3d). The broad peak centered at 3392 cm-1represents the stretching vibrations of O–H. The peaks at 2937 and 2887 cm-1attribute to stretching vibration of CH3 and CH2. And the peak at 1740 cm-1 attribute to the stretching vibration of C=O in carboxyl. A sharp absorptionpeak at 1670 cm-1 9

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associated with C=O stretching isobserved. In addition, the peaks at 1540 and 1457 cm-1can beattributed to the bending vibration of N-H and C-N. And the peaks centered at 1249 and 1054 cm-1are derived from the stretching vibration of C-O-C and C-O. The C-dots consist of nitrogen doped amorphous core functionalized with various oxygen and nitrogen related groups, such as carbonyl, carboxyl, amide, ether and C-O. 3.5 The PL properties of C-dots Fig.4a shows that the C-dots in aqueous solution have two typical UV-Vis absorption peaks at 230 and 344 nm,respectively. The peak at230 nm, corresponding tothe π-π* transition of the aromatic sp2 domains, leads to nearly no observed PL signal.

The other transition centeredat 344 nm due tothe trapping of

excitedstateenergy by the surfacestates results in strongemission. The optimal excitation and emission wavelength are at 340 and 425 nm (Fig. 4a). Very brightblue color under the excitation ofUV (365 nm) light ata low concentration (100 µg·mL-1) of

the

C-dots

aqueous

solution

canbe

clearly

seen

in

the

insetof

Fig.4a.Excitation-dependent PL behavior is observed, which is common in fluorescent carbon nanomaterials33. The emission wavelength shifts from 422 to 434 nm with the excitation wavelength increases from 300 to 390 nm (Fig 4b). The QY of C-dots is determined to be 54 % by selecting the quinine sulfate as standard34using slope method. The stability of both C-dots under various conditions has alsobeen studied. The PL properties of C-dots are pH dependent (Fig. 4c). Under acidic conditions, the PL is nearly completely quenched. When the pH is larger than 7, the PL intensity obviously increases, especially when the pH is between 7 and 9, the PL intensity dramatically increases and exhibits linear relationship. It means that the as-prepared C-dots can be used as sophisticated pH sensor to detect pH values between 7 and 9. The effect of the pH values canbe understood in terms of the change in surface charge owingto protonation–deprotonation.There was no changes in PL intensity at high ionic strengths, which issignificant because it is essential for C-dots to be used in thepresence of physical salt concentrations in practical applications (Fig. 4d). In 10

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addition, the lifetime of C-dots is 13.26 ns, which is single exponential decay (Fig. S4). This suggests that the origin of PL is one single species. 3.6 Protective effect against oxidative stress The biocompatibility of C-dots was studied by the MTT assay. As shown in Fig. S5, MTT assay demonstrated that the C-dots exhibit low cytotoxicity to SGC-7901 and GES-1, even at high concentrations (400 µg·mL-1). The protective effects of C-dots against oxidative stress on cells were then investigated. The cells were incubated with different concentrations of C-dots for 24 h and then treated with 2 mM H2O2 for 2 h. Finally the cell viability was determined by MTT assay. As shown in Fig. 5, decreased cell viability can be observed for H2O2-treated cells, which indicates the H2O2induced oxidative effect. The cell viability of SGC-7901 increased from 46% to 71% with the concentration of C-dots increased from 0 to 175µg·mL-1. Meanwhile, when the concentration of C-dots increased from 0 to 300 µg·mL-1, the cell viability of GES-1 increased from 28% to 43%.It shows thatC-dots can protect cell from H2O2induced oxidative stress and the protective effect shows a significant concentration dependent feature. Interestingly, C-dots present different protect abilities on different cell types. Some cell types have higher metabolic activities than others, and these differences can translate into higher rates of mitochondrial ROS formation. Previous results also indicate that cancer cells produce more ROS than normal cells35. We suspect thatC-dots can quench the intracellular ROS directly and lead to an increase of cell viability, or they can actively stimulate the intracellular SOD production, then protect cells from oxidative induced stress. 3.7 Protective Mechanism We used SGC-7901 as example to investigate the possible protectivemechanism of C-dots against oxidative induced stress. DCFH-DA itself couldn’t emit fluorescence. Once it goes into cells, it could behydrolyzed by enzyme in cells to produce DCFH. After oxidized by ROS, it couldexhibit strong fluorescence. Flow cytometry was used to detect the intracellularROS levelof SGC-7901. As shown in Fig. 6, compared with the control group (without C-dots), the fluorescence intensity 11

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of intracellularDCF decreased with the concentration of C-dots increased. These results indicate that the C-dots can efficiently quench the intracellular ROS, thereby protect cells from oxidative induced stress. Furthermore,the activity of SOD was evaluated for SGC-7901 using WST-1methods. SOD is an antioxidative enzyme that is suggested to be important to ROS elimination36-37.A previous report proposed that carbon based nanomaterials can potentially mediate the expression of geneswhose products contribute to a cell’sredox status and subsequently several rudimentary cell processes38. Thus, weanalyzed the alteration of intracellular SOD of SGC-7901 cellsincubated with different concentrations of C-dots. As shown in Fig. S5, C-dots can stimulate the production of SOD in a concentration dependent manner between 0-100µg·mL-1. The accumulation of intracellular SOD is beneficialto protect cells from the subsequent oxidative stress.In one word, the C-dots can eliminate intracellularROS andstimulate the intracellular SOD production to protect cell against oxidative stress.

4. Conclusions A series of C-dots using different reaction materials have been synthesized and their PL properties have been characterized. We have also summarized several basic principles to obtain highly PL C-dots. Highly PL (QY=54%) nitrogen doped C-dots have been prepared through one-step carbonizing CA and Tris and using oleic acid as solvent. The synthesized C-dots are monodisperse with narrow sizedistributionwith pH dependent PL properties and high stability in strong ionic strengths. The C-dots also exhibited protective effect against oxidativeinduced stress, which was promising for expanding the field of biological application of C-dots, especially the field of medical treatment. The protective mechanism wasproposed that C-dots can eliminate intracellularROS andstimulate the intracellular SOD production. Associated Content Supporting Information Additional information on reaction conditions for the preparation of other C-dots, effects of CA/Tris ratio and reaction time on the QY, size distribution of C-dots, XRD 12

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pattern for the C-dots, FL decays curves of the C-dots, viability of the cells after incubation with C-dots, total intracellular SOD incubated C-dots. Acknowledgments The authors gratefully acknowledge the financial support from National Science Fund for Distinguished Young Scholars of China (Grant No. 21225313), National Natural Science Foundation of China (Grant No. 21303126, 21473125),Fundamental Research Funds for the Central Universities (No.2042014kf0287). References 1.

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Reaction. Nanoscale 2013,5, 3289-3297 15.Li, H.; He, X.; Kang, Z.; Huang, H.; Liu, Y.; Liu, J.; Lian, S.; Tsang, C. H. A.; Yang, X.; Lee, S. T., Water ‐ Soluble Fluorescent Carbon Quantum Dots and Photocatalyst Design. Angew. Chem. Int. Ed.2010,49, 4430-4434. 16. Mirtchev, P.; Henderson, E. J.; Soheilnia, N.; Yip, C. M.; Ozin, G. A., Solution Phase Synthesis of Carbon Quantum dots as Sensitizers for Nanocrystalline TiO2 solar cells. J. Mater. Chem. 2011,22, 1265-1269. 17. Li, H.; Kang, Z.; Liu, Y.; Lee, S.-T., Carbon Nanodots: Synthesis, Properties and Applications. J.Mater Chem2012,22, 24230-24253. 18. Zhao, Q.-L.; Zhang, Z.-L.; Huang, B.-H.; Peng, J.; Zhang, M.; Pang, D.-W., Facile Preparation of Low Cytotoxicity Fluorescent Carbon Nanocrystals by Electrooxidation of Graphite. Chem. Commun.2008, 5116-5118. 19. Pan, D.; Zhang, J.; Li, Z.; Wu, C.; Yan, X.; Wu, M., Observation of pH-, Solvent-, Spin-, and Excitation-dependent Blue Photoluminescence from Carbon Nanoparticles. Chem. Commun.2010,46, 3681-3683. 20. Liu, H.; Ye, T.; Mao, C., Fluorescent Carbon Nanoparticles Derived from Candle Soot. Angew. Chem. Int. Ed.2007,46, 6473-6475. 21. Wang,

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Figure 1. A synthetic route using CA and Tris: from condensation to polymerization, and carbonization. Figure 2.TEM images, high-resolution TEM images of C-dots Figure 3. XPS spectra, High resolution C1s, N1s XPS, and FTIR spectra of C-dots. Figure 4.The PL properties of C-dots Figure 5. Cytoprotective effects of the C-dots against H2O2-induced stress on cells Figure 6. The ROS level in SGC-7901 detected by Flow cytometry.

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Figure 1. A synthetic route using CA and Tris: from condensation to polymerization, and carbonization.

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Figure 2 TEM images (a), high-resolution TEM images (b) of C-dots.

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Figure 3 XPS spectra (a), High resolution C1s (b), N1s XPS (c) and FTIR spectra (d) of C-dots.

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Figure 4 (a)Absorption and optimal emission spectra of C-dots. (b) Excitation dependent emission spectra of C-dots. (c) The influence of pH values on the PL intensity of C-dots. (d) Effect of ionic strengths on the PL intensity of C-dot (ionic strengths were controlled by various concentrations of KCl).

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Figure 5 Cytoprotective effects of the C-dots against H2O2-induced stress on SGC-7901 (a) and GES-1 (b) cells(mean ± SD,n = 4).

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Figure 6. (a) The ROS level in SGC-7901 detected by Flow cytometry. (b) The FL intensity of DCFH relative to the concentration of C-dots.

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A synthetic route using CA and Tris. Cytoprotective effects of the C-dots against H2O2-induced stress on cells 50x35mm (300 x 300 DPI)

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