DOI: 10.1002/slct.
201600216 Full Papers
z Materials Science inc. Nanomaterials & Polymers
Synthesis of Cellulose-Based Carbon Dots for Bioimaging
Peilian Shen, Junkuo Gao, Jingkun Cong, Ziwei Liu, Changqing Li, and Juming Yao*[a]
Photoluminescent carbon dots (CDs) as a novel carbon-based
material have attracted much more attention. Here, we re-
ported the synthesis of cellulose-based carbon dots by a facile
and highly reproducible new rote from cellulose and urea. The
as-obtained CDs were characterized via FTIR, XPS, TEM, UV-vis
and PL spectra. The as-obtained CDs showed high nitrogen
content, stable and excitation-independent blue-green photo-
Introduction
In recent years, photoluminescent carbon dots (CDs) as a novel
carbon-based material have attracted much more attention in-
creasingly, since they were reported by Xu et al in 2004.[1] Usu-
ally, CDs show good solubility, stable photoluminesce (PL), low
cytotoxicity and excellent biocompatibility. CDs have a wide
range of potential applications, such as bioimaging ion de-
tection, fluorescent printing ink, light emitting diodes (LED),
photocatalysts and so on.[2] Particularly, bioimaging and ion de-
tective application of CDs are mostly studied, in which a high
quantum yield of PL is quite essential. So as to further improve
their PL properties, numerous methods have been used includ-
ing adopting surface passivated reagents and heteroatoms
doping.[2b, c, 3] Among of them, introduction of nitrogen (N) at-
tracted much attention which exhibits a more significant effect
in carbon framework and increase nitrogen-containing group
to improve the fluorescent brightness.[4] Nitrogen doping can
effectively tune the electronic properties and surface and local
chemical relativities of CDs.[5] Thus N-doped CDs can possess
much more and effective applications. Up to now, researchers
have developed several strategies to prepare CDs, such as arc-
discharged soot, laser ablation carbon materials, electro-
chemical method, microwave-assisted polyol, hydrothermal
treatment of orange juice, citric acid and so on.[1, 6] However,
these methods suffer from some drawbacks, involving complex
processes, expensive, and severe condition. So a simple, effi-
cient and low-cost method to prepare CDs is extremely urgent.
Cellulose is the most abundant natural polymer, it exhibits
low cost, non-toxicity, biodegradability, eco-friendly, and high
[a] P. Shen, Prof.Dr. J. Gao, J. Cong, Z. Liu, C. Li, Prof.Dr. J. Yao
The Key laboratory of Advanced Textile Materials and Manufacturing
Technology of Ministry of Education, National Engineering Lab for Textile
Fiber Materials and Processing Technology (Zhejiang), College of Materials
and Textiles
Zhejiang Sci-Tech University
Hangzhou 310018, P. R. China
E-mail: yaoj@zstu.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/slct.201600216
ChemistrySelect 2016, 1, 1314 – 1317
luminesce. The quantum yield (QY) of the synthesized CDs is
up to 21.7 %. Furthermore, the low cytotoxicity and great bio-
compatibility of CDs were proved via CCK-8 assay and fluores-
cence bioimaging. The results indicate that the cellulose-based
CDs show promising applications for bioimaging in living cell
systems.
stability.[7] The advantages of cellulose make it successfully
been applied in many research areas, including water purifica-
tion, medicine, biology, nano-science, and so on.[8] Though
many biomass materials including chitosan, proteins and gela-
tin[9] have been used as raw stuff of carbon dots, so far there
are few reports about cellulose-based carbon dots.
In this work, we adopt one-step in situ hydrothermal meth-
od to synthesize N-doped CDs from cellulose for the first time
and urea used as a source of nitrogen. Compared to most of
previous synthesis methods that involve uneconomical raw ma-
terials or complex processes, it is usually cheap, facile and eco-
friendly. At the same time, the excitation-dependent N-doped
cellulose-based CDs showed comparatively high nitrogen con-
tent and high photoluminescent quantum yield of 21.7 %. The
as-prepared CDs can sever as a very effective bioimaging re-
agent.
Results and Discussion
Optimization of synthesis conditions
In this work, we designated cellulose as the carbonic raw for
fabricating CDs, while urea was used as the source of nitrogen.
Although the comprehensive mechanism of CDs’ formation is
still unclear, it involves decomposing, carbonization of stocks,
dehydration condensation, nucleation and growth of carbon in
the progress of hydrothermal experiments.[2m, 10] Meanwhile, we
studied the factors which could affect the photoluminescent
property of CDs. Herein we focus on investigating effect of
urea/cellulose ratio, temperature and reaction time. As the car-
bon source, the mass of cellulose was fixed to be 0.5 g in all
experiments. The mass of urea was modulated from 0.05 g to
0.5 g. As shown in Figure 1a, the result revealed that optimum
mass of urea was 0.35 g with a urea/cellulose weight ratio of
0.7. When the mass of urea was above or below 0.35 g, the QY
of CDs became obviously decreased. As the mass of urea in-
creased from zero, much more nitrogen was doped into the
CDs, which could improve the QY of CDs as reported in liter-
atures. Yet, when the N content is too high in CDs, we believe
that too much nitrogen would wrap the CDs and reduce the
1314  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Quantum yield (QY) of CDs as a function of the urea mass (a); re-
action time (b) with the mass of cellulose fixed to 0.5 g in all reactions.
effective surface defects, result in lower QY.[11] The influence of
reaction time was also investigated, and the time was set at
12 h, 24 h, 48 h, 72 h, and 96 h. Figure 1b displayed the influ-
ence of reaction time for the CDs obtained with urea/cellulose
weight ratio of 0.7. The reaction time of 72 h showed the high-
est QY of CDs. Then, the influence of reaction temperature was
also studied. The reaction temperature was set to 150, 180, and
200 8C, respectively. The results of CDs obtained with urea/cel-
lulose weight ratio of 0.7 and reaction time of 72 h was tabu-
lated in Table 1. The QY of CDs was increased with the rise in
Table 1. Quantum yield (QY) of CDs with urea/cellulose weight ratio of 0.7
as a function of the reaction temperature.
Temperature(8C) QY (%)
150 14.3
180 21.7
200 21.9
temperature up to 180 8C. When the reaction temperature in-
creased to 200 8C, there was no significant change of QY. From
the results, an optimum experimental condition could be sum-
marized, with a urea/cellulose weight ratio of 0.7, reaction time
of 72 h and reaction temperature of 180 8C.
Characterization
The morphology and particle size of Carbon dots synthesized
by hydrothermal process under optimal experimental con-
ditions were displayed in the transmission electron microscopy
(TEM) images. As shown in Figure 2, the CDs were quasi-spher-
Figure 2. (a) TEM image of CDs; (b) HRTEM image of CDs.
ChemistrySelect 2016, 1, 1314 – 1317
Full Papers
ical with an average diameter of 4.2 nm. High-resolution TEM
image of CDs reveal lattice fringes with interplanar spacing
0.32 nm which corresponded to the (002) diffraction facets of
graphitic carbon.[3c]
To understand the compositions and structures of CDs, the
Fourier transform infrared spectroscopy (FT-IR) and X-ray pho-
toelectron spectroscopy (XPS) were carried out. From the FT-IR
spectra of CDs (Figure 3), it revealed that the absorption bands
Figure 3. The FT-IR spectra of CDs and cellulose.
at 3408 cm 1 and 3216 cm 1 were attributed to stretching vi-
bration of O H and N H.[3c] The stretching vibration of CH3 at
2932 cm 1, stretching vibration of C=C at 1600 cm 1, stretching
vibration of C=O, C N at 1631 cm 1, and 1401 cm 1, were also
observed in the spectra.[2m, 12] Furthermore, the asymmetric
stretching modes of C O-C appeared at wavenumber of
1351 cm 1. Because the presence of N H and C N, it clearly
confirmed that urea was transformed into CDs successfully. The
XPS survey spectrum (Figure 4a) exhibited that the as-obtained
CDs mainly contain carbon, oxygen and nitrogen (C: 77.57 wt%,
N: 10.17 wt%, O: 12.27 wt%). The C1S spectrum (Figure 4b)
could be deconvoluted into 5 peaks at 284.2 eV, 284.7 eV,
285.4 eV, 286.0 eV, and 287.9 eV, which corresponded to C=C,
C C, C N, C O, and C=O groups, respectively.[3c, d, 9d, 13] The N1S
spectrum (Figure 4c) could be well-fitted with the characteristic
peaks for amino nitrogen (398.9 eV), C N bond (399.3 eV), C
N-C bond (399.7 eV), pyrrolic N (400.5 eV), indicating the in-
troduction of N elements in the CDs.[13–14] From O1s spectrum
(Figure 4d), there are 3 peaks at 531 eV, 531.6 eV and 532.6 eV,
which were assigned to C=O, C O-C and C-OH, respectively.[3c]
Optical Properties
The as-prepared CDs emerge brown and appear blue-green
fluorescence on exposure with a 365 nm UV lamp (Figure 5).
The absorption spectrum of CDs was presented in Figure 5a,
and showed a absorption peak at 272 nm, which was due to
the presence of p-p* transitions of C=C.[5b] Besides, a broad
1315  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
could greatly enhance the fluorescence intensity. Due to Nitro-
gen-doping, it resulted in forming much more N-containing
groups, which were wonderful auxochromic groups.[2n, 9d]

Cell cytotoxicity and confocal microscopy imaging

CCK-8 assay was used to evaluate the cytotoxicity of the pre-
pared CDs by using MC3T3 cells. The concentration of CDs var-
ied from 0 to 0.25 mg mL 1 for 24 h. The cells viability was
clearly seen from Figure 6, which were perceived that the cells

Figure 4. (a) XPS survey spectrum of CDs; High-resolution C1s (b), N1s (c), O1s
(d) XPS spectra of CDs.

Figure 6. Cellular toxicity results of CDs.

Figure 5. (a) UV-vis absorption spectrum and photoluminescent spectra of

CDs; inset: the photograph taken under 365 nm UV light; (b) emission spec-
tra of CDs at different excitation wavelengths from 300 to 450 nm. survival rates was still over 89 % at all experimental concen-
trations. It indicated that the CDs were not toxic and good bio-
compatible with MC3T3 cells, which could be a potential com-
petitor for application in fields of bioimaging and biosensors.
shoulder at 315 nm had been observed, probably ascribed to On account of the great photoluminescence property, narrow
n-p* transitions of C=O, and the absorption edge extends into size distribution, aqueous dispersibility, biocompatible and
the visible band.[6e] From PL excitation spectrum (Figure 5a), it non-toxic, CDs could act as a promising assay for bioimaging.
could be seen that, only a prominent peak at 330 nm was ob- In bioimaging experiments, the concentration of CDs was set
served, which was due to the p-p* transitions derived from the to 0.25 mg mL 1 with 4 h incubation, and confocal fluorescence
carbine-like triplet state of zig-zag edges of CDs.[3d] The fluo- microscope was used to testify the bioimaging performance. As
rescent emission spectrum (Figure 5a) measured with an ex- show in Figure 7, Multi-color fluorescence was distinct ob-
citation wavelength of 330 nm revealed that the emission peak
centers on 410 nm with a large stokes shift of 80 nm. The emis-
sion peaks were depended on the excitation wavelength (Fig-
ure 5b), with the increasing of excitation wavelength, the emis-
sion peaks shifted to longer wavelengths (from 410 nm to
500 nm). This phenomenon could be observed in many fluo-
rescent carbon-based materials, which was attributed to the
surface state affecting the band gap of CDs. In addition, the
surface state of CDs was similar to a molecular state; quantum
dimensions resultd in the size effect, both of which were con-
ducive to the complexity of excited states of CDs.[2n]
The highest quantum yield (QY) of the as-prepared CDs was
21.7 %, which was higher than reported CDs obtained from
Figure 7. MC3T3 cells confocal fluorescence image of CDs at excitation of
other biomass materials.[9d, 12a, 15] The high quantum yield could 405 nm (a); 488 nm (b).
be resulted from the high level of nitrogen-doping, which

ChemistrySelect 2016, 1, 1314 – 1317 1316  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

served under different excitation wavelength from cell mem-
brane and cytoplasm without the nucleus. MC3T3 cells showed
blue and green colors when they were excited with 405 and
488 nm laser pulses, respectively. All these points certified that
the CDs from cellulose and urea could serve as a promising la-
beled reagent.
Conclusions
In conclusion, cellulose-based carbon dots were fabricated by a
facile and highly reproducible new rote from cellulose and
urea. The as-obtained CDs showed high nitrogen content, sta-
ble and excitation-independent blue-green photoluminesce.
The QY of the synthesized CDs was up to 21.7 %. Furthermore,
the low cytotoxicity and great biocompatibility of CDs were
proved via CCK-8 assay and fluorescence bioimaging. The re-
sults indicate that the cellulose-based CDs show promising ap-
plications for bioimaging in living cell systems.
Acknowledgements
This work is supported by the National Natural Science Founda-
tion of China (No. 51372226 and 51402261) and Science Foun-
dation of Zhejiang Sci-Tech University (ZSTU) under Grant No.
13012138-Y. J. G. acknowledges the financial support from the
public technology research plan of Zhejiang Province, China
under Grant No. 2014C33041 and support from the Zhejiang
Provincial Top Key Academic Discipline of Textile Science and
Engineering, the Open Fund of Top priority disciplines in col-
leges and universities in Zhejiang Province,China (2015KF20).
Keywords: Carbon Dots · Cellulose · Photoluminescence ·
Bioimaging · Quantum Yield
[1] X. Xu, R. Ray, Y. Gu, H. J. Ploehn, L. Gearheart, K. Raker, W. A. Scrivens, J.
Am. Chem. Soc. 2004, 126, 12736–12737.
[2] a) J. Liu, Y. Liu, N. Liu, Y. Han, X. Zhang, H. Huang, Y. Lifshitz, S.-T. Lee, J.
Zhong, Z. Kang, Science 2015, 347, 970–974; b) S. Y. Lim, W. Shen, Z. Gao,
Chem. Soc. Rev. 2015, 44, 362–381; c) Y. Wang, A. Hu, J. Mater. Chem. C
2014, 2, 6921–6939; d) W. Wang, L. Cheng, W. Liu, Sci. China Chem. 2014,
57, 522–539; e) P. G. Luo, S. Sahu, S.-T. Yang, S. K. Sonkar, J. Wang, H.
Wang, G. E. LeCroy, L. Cao, Y.-P. Sun, J. Mater. Chem. B 2013, 1,
2116–2127; f) H. Li, Z. Kang, Y. Liu, S.-T. Lee, J. Mater. Chem. 2012, 22,
24230–24253; g) L. Cao, X. Wang, M. J. Meziani, F. Lu, H. Wang, P. G. Luo,
Y. Lin, B. A. Harruff, L. M. Veca, D. Murray, J. Am. Chem. Soc. 2007, 129,
11318–11319; h) S. N. Baker, G. A. Baker, Angew. Chem. Int. Ed. 2010, 49,
6726–6744; i) S.-T. Yang, L. Cao, P. G. Luo, F. Lu, X. Wang, H. Wang, M. J.
Meziani, Y. Liu, G. Qi, Y.-P. Sun, J. Am. Chem. Soc. 2009, 131, 11308–11309;
j) R. Liu, D. Wu, S. Liu, K. Koynov, W. Knoll, Q. Li, Angew. Chem. 2009, 121,
4668–4671; k) X. Li, S. Zhang, S. A. Kulinich, Y. Liu, H. Zeng, Sci. Rep.
2014, 4, 4976; l) S. Liu, J. Tian, L. Wang, Y. Zhang, X. Qin, Y. Luo, A. M.
Asiri, A. O. Al-Youbi, X. Sun, Adv. Mater. 2012, 24, 2037–2041; m) Q. Xu, P.
Pu, J. Zhao, C. Dong, C. Gao, Y. Chen, J. Chen, Y. Liu, H. Zhou, J. Mater.
Chem. A 2015, 3, 542–546; n) S. Zhu, Q. Meng, L. Wang, J. Zhang, Y.
Song, H. Jin, K. Zhang, H. Sun, H. Wang, B. Yang, Angew. Chem. 2013,
125, 4045–4049; o) S. Qu, X. Wang, Q. Lu, X. Liu, L. Wang, Angew. Chem.
2012, 124, 12381–12384; p) F. Wang, Y.-H. Chen, C.-Y. Liu, D.-G. Ma,
Chem. Commun. 2011, 47, 3502–3504; q) L. Cao, S. Sahu, P. Anilkumar,
ChemistrySelect 2016, 1, 1314 – 1317
Full Papers
C. E. Bunker, J. Xu, K. S. Fernando, P. Wang, E. A. Guliants, K. N. Tackett, Y.-
P. Sun, J. Am. Chem. Soc. 2011, 133, 4754–4757.
[3] a) D. Sun, R. Ban, P.-H. Zhang, G.-H. Wu, J.-R. Zhang, J.-J. Zhu, Carbon
2013, 64, 424–434; b) Y. Dong, H. Pang, H. B. Yang, C. Guo, J. Shao, Y. Chi,
C. M. Li, T. Yu, Angew. Chem. Int. Ed. 2013, 52, 7800–7804; c) H. Nie, M. Li,
Q. Li, S. Liang, Y. Tan, L. Sheng, W. Shi, S. X.-A. Zhang, Chem. Mater. 2014,
26, 3104–3112; d) A. Ananthanarayanan, Y. Wang, P. Routh, M. A. Sk, A.
Than, M. Lin, J. Zhang, J. Chen, H. Sun, P. Chen, Nanoscale 2015, 7,
8159–8165; e) W. Chen, K. Long, H. Yu, Y. Tan, J. S. Choi, B. A. Harley, B.
Cunningham, Analyst, 2014, 139, 5954–5963, f) Z. Chen, Y. Wang, Y. Yang,
N. Qiao, Y. Wang, Z. Yu, Nanoscale, 2014, 6, 14708–14715.
[4] a) R. Zhang, W. Chen, Biosens. Bioelectron. 2014, 55, 83–90; b) Z. Yang, M.
Xu, Y. Liu, F. He, F. Gao, Y. Su, H. Wei, Y. Zhang, Nanoscale 2014, 6,
1890–1895; c) D. Qu, M. Zheng, P. Du, Y. Zhou, L. Zhang, D. Li, H. Tan, Z.
Zhao, Z. Xie, Z. Sun, Nanoscale 2013, 5, 12272–12277; d) W. Li, Z. Zhang,
B. Kong, S. Feng, J. Wang, L. Wang, J. Yang, F. Zhang, P. Wu, D. Zhao,
Angew. Chem. Int. Ed. 2013, 52, 8151–8155; e) Y. Xu, M. Wu, Y. Liu, X. Z.
Feng, X. B. Yin, X. W. He, Y. K. Zhang, Chem. Eur. J. 2013, 19, 2276–2283.
[5] a) L. Wang, H. S. Zhou, Anal. Chem. 2014, 86, 8902–8905; b) X. Teng, C.
Ma, C. Ge, M. Yan, J. Yang, Y. Zhang, P. C. Morais, H. Bi, J. Mater. Chem. B
2014, 2, 4631–4639; c) S. Gao, Y. Chen, H. Fan, X. Wei, C. Hu, L. Wang, L.
Qu, J. Mater. Chem. A 2014, 2, 6320–6325.
[6] a) B. De, N. Karak, Rsc Adv 2013, 3, 8286–8290; b) J. Zhou, C. Booker, R.
Li, X. Zhou, T.-K. Sham, X. Sun, Z. Ding, J. Am. Chem. Soc. 2007, 129,
744–745; c) Y.-P. Sun, B. Zhou, Y. Lin, W. Wang, K. S. Fernando, P. Pathak,
M. J. Meziani, B. A. Harruff, X. Wang, H. Wang, J. Am. Chem. Soc. 2006,
128, 7756–7757; d) Y. Liu, N. Xiao, N. Gong, H. Wang, X. Shi, W. Gu, L. Ye,
Carbon 2014, 68, 258–264; e) S. Hu, A. Trinchi, P. Atkin, I. Cole, Angew.
Chem. Int. Ed. 2015, 54, 2970–2974; f) V. Strauss, J. T. Margraf, C. Dolle, B.
Butz, T. J. Nacken, J. Walter, W. Bauer, W. Peukert, E. Spiecker, T. Clark, J.
Am. Chem. Soc. 2014, 136, 17308–17316.
[7] a) S. Zhu, Y. Wu, Q. Chen, Z. Yu, C. Wang, S. Jin, Y. Ding, G. Wu, Green
Chem. 2006, 8, 325–327; b) C. Ye, S. T. Malak, K. Hu, W. Wu, V. V. Tsukruk,
ACS Nano 2015, 9, 10887–10895; c) Q. Yang, T. Saito, L. A. Berglund, A.
Isogai, Nanoscale 2015, 7, 17957–17963.
[8] a) H. Zhu, Y. Zhang, X. Yang, H. Liu, X. Zhang, J. Yao, Ind. Eng. Chem. Res.
2015, 54, 2825–2829; b) H. Zhu, Y. Zhang, X. Yang, H. Liu, L. Shao, X.
Zhang, J. Yao, J. Hazard. Mater. 2015, 296, 1–8; c) H.-Y. Yu, G.-Y. Chen, Y.-
B. Wang, J.-M. Yao, Cellulose 2015, 22, 261–273; d) L. Liu, Z. Y. Gao, X. P.
Su, X. Chen, L. Jiang, J. M. Yao, Acs Sustain. Chem. Eng. 2015, 3, 432–442;
e) H. Yu, B. Sun, D. Zhang, G. Chen, X. Yang, J. Yao, J. Mater. Chem. B
2014, 2, 8479–8489; f) A. W. Carpenter, C.-F. de Lannoy, M. R. Wiesner, En-
viron. Sci. Technol. 2015, 49, 5277–5287; g) P. Thoniyot, M. J. Tan, A. A.
Karim, D. J. Young, X. J. Loh, Adv. Sci. 2015, 2, 1400010; h) H.-W. Liang, Z.-
Y. Wu, L.-F. Chen, C. Li, S.-H. Yu, Nano Energy 2015, 11, 366–376; i) F. Fu, L.
Li, L. Liu, J. Cai, Y. Zhang, J. Zhou, L. Zhang, Acs Appl. Mater. Inter. 2015,
7, 2597–2606.
[9] a) Q. Liang, W. Ma, Y. Shi, Z. Li, X. Yang, Carbon 2013, 60, 421–428; b) C.
Ding, A. Zhu, Y. Tian, Acc. Chem. Res. 2013, 47, 20–30; c) Y. Yang, J. Cui,
M. Zheng, C. Hu, S. Tan, Y. Xiao, Q. Yang, Y. Liu, Chem. Commun. 2012, 48,
380–382; d) Z. L. Wu, P. Zhang, M. X. Gao, C. F. Liu, W. Wang, F. Leng, C. Z.
Huang, J. Mater. Chem. B 2013, 1, 2868–2873; e) C. Zhu, J. Zhai, S. Dong,
Chem. Commun. 2012, 48, 9367–9369.
[10] S. Sahu, B. Behera, T. K. Maiti, S. Mohapatra, Chem. Commun. 2012, 48,
8835–8837.
[11] J. Wang, P. Zhang, C. Huang, G. Liu, K. C.-F. Leung, Y. X. J. Wng, Lang-
muir 2015, 31, 8063–8073.
[12] a) S. Han, H. Zhang, J. Zhang, Y. Xie, L. Liu, H. Wang, X. Li, W. Liu, Y. Tang,
Rsc Adv 2014, 4, 58084–58089; b) X. Feng, Y. Jiang, J. Zhao, M. Miao, S.
Cao, J. Fang, L. Shi, Rsc Adv 2015, 5, 31250–31254.
[13] K. Jiang, S. Sun, L. Zhang, Y. Lu, A. Wu, C. Cai, H. Lin, Angew. Chem. Int.
Ed. 2015, 54, 5360–5363.
[14] L. Wang, Y. Yin, A. Jain, H. S. Zhou, Langmuir 2014, 30, 14270–14275.
[15] J. Zhang, Y. Yuan, G. Liang, S. H. Yu, Adv. Sci. 2015, 2, 1500002.
Submitted: March 17, 2016
Accepted: April 26, 2016
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