Green synthesis of nitrogen-doped carbon dots from ginkgo fruits and the application in cell imaging

Accepted Manuscript Green synthesis of nitrogen-doped carbon dots from ginkgo fruits and the application in cell imaging

Lingling Li, Luyao Li, Chang-Po Chen, Fengling Cui PII: DOI: Reference:

S1387-7003(17)30530-0 doi:10.1016/j.inoche.2017.10.006 INOCHE 6794

To appear in:

Inorganic Chemistry Communications

Received date: Revised date: Accepted date:

1 July 2017 7 October 2017 8 October 2017

Please cite this article as: Lingling Li, Luyao Li, Chang-Po Chen, Fengling Cui , Green synthesis of nitrogen-doped carbon dots from ginkgo fruits and the application in cell imaging. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Inoche(2017), doi:10.1016/j.inoche.2017.10.006

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ACCEPTED MANUSCRIPT Green synthesis of nitrogen-doped carbon dots from ginkgo fruits and the application in cell imaging Lingling Lia,b, Luyao Lia , Chang-Po Chena,*, Fengling Cuia,* a

College of Chemistry and Chemical Engineering, Key Laboratory of Green

Chemical Media and Reactions, Ministry of Education, National Demonstration

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Center for Experimental Chemistry Education, Henan Normal University, Xinxiang 453007, China

Department of Medicine, Hebi Polytechnic, Hebi 458030, China

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b

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Abstract

In the report, microwave-assisted and hydrothermal methods were adopted

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respectively to produce nitrogen doped carbon dots (N-CDs) using ginkgo fruits as the sole carbon source without additional surface passivation agents. The differences

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between these two N-CDs were compared in luminescence properties, particle sizes and morphologies. The TEM images showed that both microwave-assisted N-CDs

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(M-N-CDs) and hydrothermal N-CDs (H-N-CDs) were well dispersed, and the

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morphology of H-N-CDs was more uniform. The fluorescence results indicated the H-N-CDs possessed better fluorescence emission characteristic. Thus, the H-N-CDs with better luminescence properties were characterized by FT-IR, XPS, fluorescence

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and UV-Vis spectroscopy. Also, the stability and cytotoxicity of H-N-CDs synthesized by ginkgo fruits as the fluorescent probes were studied. Eventually, the H-N-CDs

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were applied in cell imaging using HeLa cells and KYSE410 cells as the cell models. Keywords: CDs; Microwave-assisted method; Hydrothermal method; Cell imaging

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Corresponding authors: Tel/Fax: +86-373-3326336. E-mail: [email protected], [email protected]

ACCEPTED MANUSCRIPT 1. Introduction On the way of seeking eco-friendly and mild fluorescent nanomaterials with good biocompatibility, carbon dots (CDs) have been considered as the promising candidates to replace the traditional semiconducting quantum dots[1-3]. CDs have attracted enormous attention since the discovery, due to their unique and superior properties

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such as tunable excitation and emission spectra, high photostability, aqueous solubility, and low toxicity. Because of their outstanding merits, CDs have been developed and

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applied in many potential fields, such as fluorescence imaging, photoacoustic imaging

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and drug delivery[4-8],sensors[9], catalysis[10]. Recently, a variety of methods have been developed for the synthesis of CDs, such as microwave radiation, hydrothermal

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treatment, laser ablation, arc discharge, oxidation and so on. Among these methods, laser ablation and arc discharge are limited by using expensive, complicated and

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energy-intensive equipment. And chemical oxidation needs to rely on strong acid. Microwave radiation and hydrothermal treatment are two common methods to synthesize CDs. Microwave radiation method requires short time and the reaction

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procedure could be completed in few minutes due to the high energy. Hydrothermal

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method is simple and possesses the advantage of low-cost and manageable reaction condition. In spite of great progress achieved in the synthesis field, the more extensive

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application of CDs is limited by harsh reaction conditions, the toxic precursors, complicated subsequent surface passivation procedure and the other restrictive factors.

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The green synthesis of CDs using natural renewable carbon source is an attractive research subject. Because the carbon source is cheap, clean, nontoxic and accessible, exploring green synthesis method of CDs has attracted extensive attention. And some researches have reported using biological precursors, such as grape juice[11], milk[12], potatoes[13], melanin[14], glycine[15], oats[16], glucose and glutamic acid[17], and even the waste biomass[18-19] to prepare CDs. However, producing large scale CDs with high quantum yields (QYs) by simple one-step method is still a critical challenge. In the paper, the one-pot green synthesis of nitrogen doped carbon dots with excellent

ACCEPTED MANUSCRIPT luminescence performance using gingko fruits as the only carbon materials were reported. The method was green, simple and low cost without additional surface passivation. We compared the advantages and disadvantages of the two products by microwave-assisted and hydrothermal treatment respectively, and found the H-N-CDs with better photoluminescence property. So the physicochemical property of

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H-N-CDs were characterized using FT-IR, XPS, fluorescence and UV-Vis spectroscopy. The fluorescence stability of H-N-CDs as fluorescence probe was

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explored. Finally, HeLa cells and KYSE410 cells were selected as the models to investigate the cytotoxicity and imaging application of H-N-CDs.

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2. Experiment

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2.1 Synthesis of N-CDs

Firstly, gingko fruits were washed clean for juicing. Then the juice was reacted by

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microwave-assisted and hydrothermal treatment respectively. The luminescence properties of two products were characterized for comparison (Fig S1). 2.2 Cytotoxicity Assays

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The cell viability was measured using the MTT assay.

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2.3 In Vitro Fluorescence Imaging

HeLa cells and KYSE410 cells were selected to investigate the cytotoxicity and

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imaging application.

3. Results and discussions

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3.1 Fluorescence and UV-Vis spectra characterization of N-CDs Microwave-assisted and hydrothermal methods are two common methods to synthesize carbon dots. In this work, these two methods were used to obtain N-CDs respectively, and characterized the fluorescence spectra of the products. The results were showed in Fig. 1. Fig. 1 displayed the fluorescence spectra of N-CDs by microwave radiation for different time and hydrothermal treatment at different temperature. Before that, the influence at different time of the fluorescence spectra of H-N-CDs were investigated. The result (Fig. S2) indicated that fluorescence intensity of N-CDs reacting for 16 h only slightly increased comparing with that reacting for 12

ACCEPTED MANUSCRIPT h. So the reaction time of 12 h was chosen. Obviously, the two N-CDs both showed typical absorption and PL spectra. Besides, the fluorescence intensity of H-N-CDs was much higher than that by microwave irradiation. Fig. 1 showed the H-N-CDs at 200 °C with the best fluorescence intensity. In order to verify this conclusion, the quantum yields (quinine sulfate as the reference) and lifetime of two kinds of N-CDs

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were investigated. It was found that quantum yields and lifetime of the H-N-CDs synthesized at 200 °C were 3.33% and 6.16 ns respectively, while these of the

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H-N-CDs heated for 15 min were 0.65% and 4.14 ns. Then the N-CDs by hydrothermal treatment at 200 oC were chosen for further characterization and

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application. From Fig. 1f, the maximum fluorescence emission wavelength shifted with the excitation wavelength increasing in the range of 340 nm to 500 nm. And the

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maximum fluorescence intensity tended to increase first and then decrease. The fluorescence intensity of carbon dots was the largest in the 380 nm excitation. Thus,

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380 nm was set as the excitation wavelength in the following fluorescence spectra measurement. UV-Vis spectrum (Fig. S3) displayed an obvious absorption at 281 nm,

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which may be caused by the π-π* transition of carbon backbone[12].

Fig. 1 Fluorescence spectra of M-N-CDs heated for (a) 5 min (b) 10 min (c) 15 min and H-N-CDs synthesized at (a) 160 oC (b) 180 oC (c) 200 oC. 3.2 TEM characterization of N-CDs To further understand the difference on the morphology and particle size of H-N-CDs and M-N-CDs, the TEM images of the two CDs were measured. Fig. 2 was the TEM

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image and size distribution of M-N-CDs heated for 15 min under 800 W. The M-N-CDs was mainly distributed between 2 nm and 4 nm with the average size of 2.82 nm. The HRTEM image showed there was lattice spacing of 0.23 nm corresponding to the (100) lattice plane of graphitic carbon[20]. Fig. 3 was the TEM image and size distribution of H-N-CDs synthesized at 200 oC. We obtained spherical carbon dots with uniform particle from gingko fruits via hydrothermal treatment. The H-N-CDs were mainly dispersed between 2 nm and 6 nm with the average size of 3.81 nm. Lattice spacing of 0.21 nm in HRTEM image corresponded to the (100) lattice plane[19].

Fig. 2 (a) TEM image of M-N-CDs heated for 15 min under 800 W (inset:

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corresponding HRTEM image) (b) The size distribution of M-N-CDs.

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Fig. 3 (a) TEM image of H-N-CDs synthesized at 200 oC (inset: corresponding HRTEM image) (b) The size distribution of H-N-CDs. Comparing these results, it can be found that the size of M-N-CDs was smaller than H-N-CDs. However, the fluorescence property of H-N-CDs was much better than the M-N-CDs. Maybe it was because of H-N-CDs with more regular and uniform morphology, which could be easily seen from TEM images. On the other hand, it may be related to the luminescence mechanism of carbon dots, which not only be affected by the particle size, but also be in relation with the surface defects of carbon dots. 3.3 FT-IR and XPS spectra analysis of H-N-CDs

ACCEPTED MANUSCRIPT The FT-IR spectrum was used to analyze the surface functional groups of H-N-CDs. The results were exhibited in Fig. S4. The peak at 3400 cm-1 was assigned to O-H bonds[21]. The peak at 2930 cm-1 was attributed to N-H bonds[22]. The stretching vibrations of C=O were at 1718 cm-1 and 1671 cm-1[23]. The vibrations of C-C and C-O bonds appeared at 1603 cm-1 and 1127 cm-1 respectively[7, 12]. The peaks at 1204 cm-1 and 1021 cm-1 were related to C-O-C bonds[24]. The stretching vibration at

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1384 cm-1 was attributed to C-N= bonds[25].

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The surface states of H-N-CDs were also analyzed by XPS spectra. There were three main peaks at 285.0 eV, 401.0 eV, and 531.0 eV corresponding to C1s, N1s, and O1s

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in Fig. S5a, indicating nitrogen element was doped into the carbon dots successfully. The C1s peaks at 284.3 eV, 285.6 eV, 287.3 eV, and 288.3 eV shown in Fig. S5b could

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be assigned to carbon in the form of C-C, C-N, C-O and C=O respectively[8]. The O1s peaks at 531.8 eV and 533.2 eV shown in Fig. S5c were associated with C=O and

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C-OH/C-O-C[26]. The N1s peaks at 399.2 and 401.6 eV shown in Fig. S5d indicated that nitrogen existed mostly in the form of C-N-C and N-H, respectively[27]. The

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characterization results from FT-IR and XPS indicated that the surface of the

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as-synthesized H-N-CDs was functionalized by -COOH and -NH2, making the H-N-CDs with better water solubility. 3.4 Fluorescence stability of H-N-CDs

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Fig. S6 were the fluorescence intensity of H-N-CDs at different pH and ionic strength. With pH or ionic strength increasing, the maximum fluorescence intensity of

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H-N-CDs changed little. This showed that the H-N-CDs had good stability in acid or alkali environment, which added possibility in practical application. The fluorescence intensity change of H-N-CDs with the presence of some common metal ions and biological disruptors were also explored. The results (Fig. 4) showed slight change in the fluorescence intensity of H-N-CDs under the condition of 50 μM of the substances mentioned above, manifesting the fluorescence intensity of H-N-CDs were not easily be interfered by metal ions and biological disruptors. The exposure time was also affect the fluorescence intensity of H-N-CDs. After one year, the fluorescence intensity was only reduced by 25%. Above all, the H-N-CDs had strong fluorescence

ACCEPTED MANUSCRIPT intensity, good stability and were not sensitive to pH , metal ions, ionic strength and biological disruptors. Therefore, the H-N-CDs had potential for practical application

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due to their well photoluminescence performance and resistance to environment.

Fig. 4 Influence of metal ions and biological disruptors on the fluorescence intensity

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of H-N-CDs.

3.5 Cytotoxicity and cell imaging of H-N-CDs

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The cell toxicity of H-N-CDs by MTT method was tested firstly using HeLa cells as the model before investigating their biological application. Adding H-N-CDs into the

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cultured HeLa cells and incubating for 24 h, then calculating the cell viability based

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on the OD values. MTT result (Fig. S7) showed HeLa cell viability was no apparent change with increasing concentration of N-CDs. Even if the concentration of H-N-CDs reached 2000 μg/mL, HeLa cells did not appear death obviously. This

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indicated that the H-N-CDs had good biocompatibility and low cytotoxicity, and thus possessed potential in biological application.

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Afterwards, the application of H-N-CDs in cell imaging was studied. After incubating HeLa cells and KYSE410 cells with N-CDs solution for 8 h, and washing off the H-N-CDs outside the cells, the Laser Scanning Confocal Microscope were employed to capture the fluorescence images of these cells with excitation at 405 nm and 488 nm respectively. From Fig. 5, it was clear there were bright blue and green fluorescence in cells, which demonstrated the synthesized H-N-CDs could enter into the cells and achieve imaging. Besides, we found the fluorescence in nucleus was weak, which indicated the H-N-CDs mainly entered into cytoplasm. The imaging results confirmed the H-N-CDs from gingko fruits were fit for biological label as

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fluorescence probes.

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Fig. 5 Laser scanning confocal microscopy images of HeLa cells at (a) 405 nm (b) 488 nm excitation and KYSE410 cells at (c) 405 nm (d) 488 nm excitation labeled with H-N-CDs. Scale Bar: 30 μm.

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4. Conclusion

In summary, two kinds of carbon dots by microwave-assisted and hydrothermal

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methods from gingko fruits were obtained and the differences on the morphology,

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particle size and photoluminescence property were compared. The carbon dots through hydrothermal treatment with better luminescence performance were characterized using FT-IR, XPS, TEM, fluorescence and UV-Vis spectroscopy. The

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results showed the hydrothermal product was with regular shape and uniform size distribution. The MTT experiments showed that H-N-CDs had stable fluorescence

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property, well biocompatibility and low toxicity. Meanwhile, cell imaging experiment showed that the N-CDs from gingko fruits by hydrothermal approach were excellent biological imaging reagents, and have the potential for practical application.

Acknowledgment The supports from the Program for Innovative Research Team in University of Henan Province (18IRTSTHN003), National Natural Science Foundation of China (30970696) and Instrumental Analysis Center of Tsinghua University are greatly appreciated.

ACCEPTED MANUSCRIPT Reference [1]

S. N. Baker, G. A. Baker, Luminescent carbon nanodots: emergent nanolights, Angew. Chem. Int. Edit. 49 (2010) 6726-6744.

[2]

H. Li, Z. Kang, Y. Liu et al, Carbon nanodots: synthesis, properties and applications, J. Mater. Chem. 22 (2012) 24230-24253. L. Cao, S. T. Yang, X. Wang, et al, Competitive performance of carbon

PT

[3]

“quantum” dots in optical bioimaging, Theranostics. 2 (2012) 295-301. W. Xiao, Y. Li, C Hu, et al, Melanin-originated carbonaceous dots for triple

RI

[4]

SC

negative breast cancer diagnosis by fluorescence and photoacoustic dual-mode imaging, J. Colloid. Interf. Sci. 497 (2017) 226-232.

S. Ruan, J. Chen, X. Cun et al, Noninvasive in vivo diagnosis of brain glioma

NU

[5]

using RGD-decorated fluorescent carbonaceous nanospheres, J. Biomed.

[6]

MA

Nanotechnol. 11 (2015) 2148-2157.

J. Chen, X. Cun, S. Ruan et al, Glioma cell-targeting doxorubicin delivery and redox-responsive

release

using

angiopep-2

decorated

carbonaceous

Ruan S, Qian J, Shen S, et al. Fluorescent carbonaceous nanodots for

PT E

[7]

D

nanodots[J]. Rsc Adv, 5 (2015) 57045-57049.

noninvasive glioma imaging after angiopep-2 decoration[J]. Bioconjugate

[8]

CE

chemistry, 2014, 25(12): 2252-2259. Q. Wang, X. Huang, Y. Long et al, Hollow luminescent carbon dots for drug

[9]

AC

delivery, Carbon. 59 (2013) 192-199. X. Gong, W. Lu, M. C. Paau et al, Facile synthesis of nitrogen-doped carbon dots for Fe(3+) sensing and cellular imaging, Anal. Chim. Acta. 861 (2015) 74-84. [10]

P. Mondal, K. Ghosal, S. K. Bhattacharyya et al, Formation of a gold–carbon dot nanocomposite with superior catalytic ability for the reduction of aromatic nitro groups in water, Rsc. Adv. 4 (2014) 25863-25866.

[11]

H. Huang, Y. Xu, C. J. Tang et al, Facile and green synthesis of photoluminescent carbon nanoparticles for cellular imaging, New. J.Chem. 38

ACCEPTED MANUSCRIPT (2014) 784. [12]

L. Wang, H. S. Zhou, Green synthesis of luminescent nitrogen-doped carbon dots from milk and its imaging application, Anal. Chem. 86 (2014) 8902-8905.

[13]

V. N. Mehta, S. Jha, R. K. Singhal et al, Preparation of multicolor emitting carbon dots for HeLa cell imaging, New. J. Chem. 38 (2014) 6152-6160. C. Hu, Y. Liu, J. Chen et al, A simple one-step synthesis of melanin-originated

PT

[14]

red shift emissive carbonaceous dots for bioimaging, J. Colloid. Interf. Sci.

[15]

RI

480 (2016) 85-90.

S. Ruan, J. Qian, S. Shen et al, A simple one-step method to prepare

SC

fluorescent carbon dots and their potential application in non-invasive glioma imaging, Nanoscale. 6 (2014) 10040-10047.

L. Shi, X. Li, Y. Li et al, Naked oats-derived dual-emission carbon nanodots

NU

[16]

for ratiometric sensing and cellular imaging, Sensor. Actuat. B-Chem. 210

[17]

MA

(2015) 533-541.

S. Ruan, J. Qian, S. Shen et al, Non-invasive imaging of breast cancer using

[18]

PT E

(2015) 25428-25436.

D

RGDyK functionalized fluorescent carbonaceous nanospheres[J]. Rsc. Adv. 5

Y. Hu, J. Yang, J. Tian et al, Waste frying oil as a precursor for one-step synthesis of sulfur-doped carbon dots with pH-sensitive photoluminescence,

[19]

CE

Carbon. 77 (2014) 775-782. D. Sun, R. Ban, P. H. Zhang et al, Hair fiber as a precursor for synthesizing of

AC

sulfur- and nitrogen-co-doped carbon dots with tunable luminescence properties, Carbon. 64 (2013) 424-434. [20]

Y. Dong, H. Pang, H. B. Yang et al, Carbon-based dots co-doped with nitrogen and sulfur for high quantum yield and excitation-independent emission, Angew. Chem. Int. Edit. 52 (2013) 7800-7804.

[21]

S. Qu, X. Wang, Q. Lu et al, A Biocompatible Fluorescent Ink Based on Water ‐Soluble Luminescent Carbon Nanodots, Angew. Chem. Int. Edit. 124 (2012) 12381-12384.

[22]

S. Liu, J. Tian, L. Wang et al, Hydrothermal treatment of grass: a low-cost,

ACCEPTED MANUSCRIPT green route to nitrogen-doped, carbon-rich, photoluminescent polymer nanodots as an effective fluorescent sensing platform for label-free detection of Cu(II) ions, Adv. Mater. 24 (2012) 2037-2041. [23]

C. Wang, Z. Xu, H. Cheng et al, A hydrothermal route to water-stable luminescent carbon dots as nanosensors for pH and temperature, Carbon 82

[24]

PT

(2015) 87-95. V. N. Mehta, S. Jha, H. Basu et al, One-step hydrothermal approach to

RI

fabricate carbon dots from apple juice for imaging of mycobacterium and fungal cells, Sensor. Actuat. B-Chem. 213 (2015) 434-443.

K. Jiang, S. Sun, L. Zhang et al, Red, green, and blue luminescence by carbon

SC

[25]

dots: full-color emission tuning and multicolor cellular imaging, Angew.

[26]

NU

Chem. Int. Edit. 54 (2015) 5360-5363.

H. Nie, M. Li, Q. Li et al, Carbon Dots with Continuously Tunable Full-Color

MA

Emission and Their Application in Ratiometric pH Sensing, Chem. Mater. 26 (2014) 3104-3112.

D

M. Xu, G. He, Z. Li et al, A green heterogeneous synthesis of N-doped carbon

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dots and their photoluminescence applications in solid and aqueous states,

CE

Nanoscale. 6 (2014) 10307-10315.

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[27]

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Graphical Abstract

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The differences between two kinds of N-CDs synthesized by microwave-assisted and hydrothermal methods were compared, and the H-N-CDs was applied in cell imaging.

ACCEPTED MANUSCRIPT Highlights The methods were green, simple and low cost without additional surface passivation. The carbon source is cheap, clean, nontoxic and accessible. The N-CDs synthesized had strong luminescence intensity and low cytotoxicity.

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The N-CDs were proper for imaging in cells or vivo as fluorescence probe.