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Available at www.sciencedirect.com

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journal homepage: www.elsevier.com/locate/carbon

Multifunctional carbon dots with high quantum

yield for imaging and gene delivery
Liming Hu a,*, Yun Sun a, Shengliang Li b, Xiaoli Wang a, Kelei Hu b, Lirong Wang b,
Xing-jie Liang b, Yan Wu b,*
a
b

College of Life Science and Bioengineering, Beijing University of Technology, No. 100, Pingleyuan, Chaoyang, Beijing 100124, China
National Center for Nanoscience and Technology, No. 11, First North Road, Zhongguancun, Beijing 100190, China

A R T I C L E I N F O

A B S T R A C T

Article history:

Carbon dots (CD) are luminescent nanomaterial with unique properties that show great

Received 5 June 2013

potential in many applications. Herein, branched polyethyleneimine-based carbon dots

Accepted 10 October 2013

(PCD) are prepared from branched polyethyleneimine by oxidation and a modified hydro-

Available online 24 October 2013

thermal reaction. Structure and composition analysis indicate that obtained PCD possesses
a 34 nm in diameter and a graphitic structure with lattice spacing of 0.30 nm. The PCD has
a quantum yield of 54.3%. The bright photoluminescence shows that it can be used for cell
imaging. The PCD exhibits extremely good biocompatibility and can be applied for gene
delivery. Because of its specific nanostructure and photoluminescence property, the multifunctional PCD prepared shows potential for applications in bioimaging and gene delivery.
 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

As a new class of fluorescent nanomaterial with the sizes below 10 nm [1], carbon dots (CD) are attracting immense attention owing to the favorable optical properties along with
chemical stability, nonblinking and low toxicity [25]. However, the complicated preparation methods and low quantum
yield of the reported CD limited their applications for biological labeling, cell imaging, drug and gene delivery. For instance, Sun et al. prepared fluorescent CD with more than
five synthetic steps via laser ablation of the carbon target
[5]. Yang et al. also reported a simple hydrothermal method
to obtain CD, but with a poor quantum yield of 1.3% [6].
Therefore, it was in great need to construct highly fluorescent
CD with a simple method.
Among the reported methods, surface passivation was
essential to improve the fluorescent efficiency of CD [712].
Recently, one-step hydrothermal method without the surface

passivation has been employed to construct CD [6]. However,

the absence of surface passivation resulted in the low fluorescent quantum yield. Moreover, it was reported that the oxidation state of CD played a vital role to improve its quantum
yield [13,14]. Therefore, in order to enhance the fluorescence
quantum yield, oxidation was introduced to the one-step synthesis of CD.
Branched polyethyleneimine (bPEI) has already been
widely applied for gene delivery owing to its significant positive charge and proton-sponge effect [1517]. Shen et al. has
already constructed CD based on bPEI, but showed limited
fluorescence efficiency and hardly been used as imaging
probe [18]. Herein, by employing bPEI as carbon source, we
established a simple and effective passivation-free route to
produce highly fluorescent CD, which partially preserved
the unchanged bPEI chains. Multifunctional nanoparticles of
bPEI-based carbon dots (PCD) demonstrated the potential for
cell imaging and gene delivery.

* Corresponding authors: Fax: +86 10 67396211.

E-mail addresses: huliming@bjut.edu.cn, huliming2813@sohu.com (L. Hu), wuy@nanoctr.cn (Y. Wu).
0008-6223/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbon.2013.10.023

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2.

Experimental

2.1.

Synthesis of PCD

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bPEI was firstly dissolved in distilled water with the concentrations of 0.510 mg/mL. Then the solution was treated with
1 M ammonium persulfate (APS) at different molar ratios. Finally, the pale-yellowish solution was transferred to a hydrothermal container and heated at 100200 C for 510 h. After
the above reaction, the resulted brown solution was centrifuged at 15,000 rpm to remove large particles and then the
supernatant was subjected to dialysis (Mw = 3000) against distilled water for 4 days.

2.2.

Characterization

Transmission electron microscope (TEM) and high resolution

transmission electron microscope (HRTEM) images were obtained on FEI Tecnai F20. Fourier transform infrared spectroscopy (FT-IR) spectra of the PCD were detected on a
spectrophotometer (PerkinElmer, Fremont, CA, USA) using
KBr as a sample matrix. Proton nuclear magnetic resonance
(1H NMR) and carbon nuclear magnetic resonance (13C NMR)
spectra of the PCD were obtained with a Bruker AVANCE 400
NMR spectrometer (Billerica, MA, USA). In addition, the fluorescence spectra were detected using a LS-55 Fluorescence Spectrometer (Perkin-Elmer, Fremont, CA, USA) and the
fluorescence quantum yield was determined with quinoline
sulfate as the reference. Nanosecond fluorescence lifetime
experiments were performed by the time-correlated singlephoton counting (TCSPC) system (Edinburgh Instruments
EPL375).

2.3.

Quantum yields measurements

Quinine sulfate aqueous (Quantum yield = 0.54) were chosen

as standards. The quantum yield of PCD (in water) was calculated according to
Fu As
Yu Ys 

1
Fs Au
where Y was the quantum yield, F was the measured integrated emission intensity, and A was the optical density. The
subscript s refers to standard with known quantum yield
and u for the sample. In order to minimize re-absorption effects, absorption in the 10 mm fluorescence cuvette was kept
below 0.10 at the excitation wavelength (360 nm).

2.4.

Cell imaging

For the in vitro imaging, MCF-7 cells were cultured in Dulbeccos modified eagle medium (DMEM) medium containing
PCD (6 ng/mL) for 4 h in confocal dishes. The cells were then
imaged with a laser confocal scanning microscope (Zeisis)
equipped with a 60 oil immersion lens. The excitation wavelengths were 405 and 488 nm.

2.5.

Agarose gel electrophoresis

The binding of DNA with PCD was evaluated by agarose gel

electrophoresis. The DNA/PCD complexes at different weight

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ratios were firstly mixed and then incubated for 30 min at

room temperature. After that, 2 lL of complex-containing
solution was mixed with 6 lL loading buffer, and loaded into
a 1% agarose gel containing ethidium bromide (0.5 mg/mL).
Then the DNA bands were visualized under a UV transilluminator at a wavelength of 365 nm.

2.6.

Cell viability assay

MCF-7 cells were seeded at a density of 5 103 cells per well in

96-well plates in DMEM medium and incubated for 24 h. The
medium was then replaced with 200 lL of medium containing
various equivalent concentrations of PCD and bPEI. The cells
were incubated for 48 h and cytotoxicity assays were performed using CCK-8 Kits (Dojindo Molecular Technologies, Tokyo, Japan). Absorbance was detected at 450 nm using a TECAN
Infinite M200 microplate reader (Tecan, Durham, USA). All data
were presented as mean percentages standard error of measurement (SEM) in triplicate compared to the optical density
values of untreated cells.

2.7.

In vitro transfection

In vitro transfection of PCD was assayed in 293T cells. Just

prior to transfection, the cell medium was aspirated and replaced with 450 lL of serum-free or 10% serum-containing
DMEM. DNA/PCD complexes (50 lL, containing 2 lg DNA) at
weight ratios of 5 were then added to each well. After incubating for 4 h, the complexes that were not internalized were removed by aspirating the medium and replacing with fresh
medium. The transfected cells were re-incubated for additional 48 h. After incubation, the cells were washed with
phosphate buffer solution (PBS) twice and then imaging with
a laser confocal scanning microscope (Zeiss) equipped with a
20 lens. The excitation wavelengths were 405 and 488 nm.

3.

Results and discussion

The overall synthetic procedure was illustrated in Fig. 1a.

bPEI solution was firstly oxidized to obtain oxygen-rich
starting material, after treatment of 1 M APS for 1 h. Then
the resulted yellow starting material was transferred into
the hydrothermal container and heated at 200 C for 10 h.
Finally, large carbon nanoparticles were removed by centrifuge and the light yellow supernatant was dialyzed against
distilled water to obtain highly fluorescent CD. As can been
seen from the right inset of Fig. 1a, the PCD solution demonstrated comparable fluorescence in contrast to fluorescein isothiocyanate (FITC) at the diluted solution when
irradiated by UV light at 360 nm. The molar ratios of bPEI/
APS and their reaction time were important for the construction of multifunctional CD because excessive oxidation
would disrupt the structure of bPEI while the absence of
oxidant resulted in weak fluorescence. bPEI-based CD without the pretreatment with APS were also prepared through
hydrothermal to compare its performance with PCD.
As shown in Fig. 1b and c, the as-prepared PCD were uniform, well dispersed and had a narrow size distribution with
the average diameters 34 nm (Statistical results are shown in

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Fig. 1 (a) Schematic illustration of the construction of the multifunctional PCD; (b and c) TEM (b) and HRTEM image (c) of PCD
The inset is the HRTEM image.

Fig. S1). Furthermore, HRTEM images showed that the PCD

possessed a graphitic structure with lattice spacing of
0.30 nm (upper inset in Fig. 1c), which was in close match
with the (0 0 2) lattice spacing of graphite [19]. The graphitic
structure was further confirmed by Fast Fourier Transformation (FFT) of the HRTEM image (Fig. S2). The 1H NMR and 13C
NMR spectra (Figs. S3 and S4) revealed the presence of the carbonyl groups. FT-IR spectra was also recorded to identify the
organic functional groups on PCD. Compared to the spectra
of bPEI, the new peaks at 1648 and 1301 cm1 of PCD were
attributed to the C@ O and COC that formed in the oxidation treatment of APS and hydrothermal process. However,
the spectra of PCD and bPEI demonstrated the same characteristic peaks at 3366, 1568 and 1467 cm1, which evidenced
that the PCD still preserved abundant bPEI chains out of the
carbon core (Fig. S5). In addition, the zeta potential of PCD
was 23.8 0.4 mV, which suggested the existence of amino
group and showed their potential to condense DNA as the
gene carrier.

The PCD showed significant blue fluorescence when irradiated by UV light at 360 nm at the diluted solution (upper inset
in Fig. 2a). Using quinine sulfate as a reference and 360 nm as
the excitation wavelength, the photoluminescence quantum
yield of PCD was measured to be 54.3%, which was bright enough for bioimaging applications (Table s1). This value was
comparable to that of reported quantum dots [20] and much
higher than that of previous reported CD [6,21]. The PCD solution showed the UVvis absorption at 335 nm, which was consistent with the result of graphite structure (around 320 nm).
The fluorescence spectra showed the maximum emission at
460 nm and the maximum excitation at 335 nm. The full
width at a half maximum (FWHM) at 460 nm was only
75 nm, which was smaller than those are previously reported
by the hydrothermal or microwave methods. Interestingly,
the PCD did not exhibit the excitation-dependent emission
behavior as previously reported CD [2]. When the solution of
PCD was excited at wavelengths from 325 to 445 nm, the maximum emission was still at 460 nm and no peak shift was ob-

Fig. 2 (a) UVvis absorption spectra (black line) and luminescence excitation (blue line) and emission spectra (red line); upper
inset, the aqueous solution of PCD in the bright field (left) and UV light at 360 nm (right). (b) A typical time-resolved
fluorescence-decay curve of PCD (ex = 360 nm) measured at 460 nm.

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served, which showed that there was no multi-fluorescence

center (Fig. S6). The fluorescent lifetime of the PCD was determined using a TCSPC instrument (Fig. 2b). The average lifetime was 4.8 ns and contained two components with
different ratios, 5.5 ns (80%) and 2.0 ns (20%), and no multi-electron trapping was observed. Additionally, the PCD
showed excellent photostability, and their fluorescence reserved more than 80% even after continuous excitation with
a 300 W Xe lamp for 72 h (Fig. S7). Furthermore, the PCD
was stable in a wide range of pH values (pH 39) and still demonstrated pH-dependent fluorescence, which slightly decreased with the increase of pH but showed significantly
decrease at pH 12 in the photoluminescence quantum yields
(58.4% and 46.3% for pH 2 and 12, respectively; Fig. S8). However, no shift of the emission peak was observed when exited
at 360 nm at different pH values (data not shown). This pHdependent fluorescence could be related to the protonation
of amino group of bPEI chain surrounding the PCD surface,
which may provide a hint as to the mechanism of PCD photoluminescence. The surrounding bPEI chains could be protonation at acid condition and deprotonation at alkaline
environments. Due to the chargecharge interactions, the
protonation of bPEI chains demonstrated much more compact structure than that at the alkaline environments, which
resulted in the enhanced fluorescence [22]. The bPEI-based
CD without the oxidation of APS was also prepared with same
hydrothermal treatment to determine their optical property.
Although this kind of CD without oxidation showed blue fluorescence as the PCD, its quantum yield was as low as 0.7%.
Furthermore, the quantum yield of PCD with different molar
ratios of APS/bPEI was investigated. As can be seem from
Fig. S10, the quantum yield of PCD was improved from 0.7%
to 54.3% with the increasing molar ratios from 0 to 25. How-

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ever, when the molar ratios of APS/bPEI was kept increasing

from 25 to 250, the quantum yield was dropped to 1.2%. The
possible mechanism of the enhanced fluorescence of PCD
may be in closely relation to oxidation pretreatment of bPEI.
Massimo et al. demonstrated that the CD derived from oxidized nanotubes exhibited stronger fluorescence than that
from pristine nanotubes [23]. Both Eda and Luo indicated that
the fluorescence of graphene oxide dots was quenched by
excessive reduction [13,24]. Moreover, they concluded that
the significant decrease of oxygen segment could lead to the
interconnectivity of the localized sp2 sites, thus resulted in
the nonradioactive recombination centers and poor fluorescence. In our experiment, the introduction of oxidized bPEI
may shape the oxygencarbon matrix of PCD and yield hard
band gap consistent with highly blue emission. However,
the detailed mechanism was still unclear and should be further studied.
It was firstly evaluated that the possibility of using the PCD
as fluorescence probes for cell imaging. After incubation with
PCD for 24 h, MCF-7 cell was fixed and tubulin was stained in
order to indicate the cell cytoplasm. As shown in Fig. S9, significant luminescence of the PCD inner the cells were observed using excitation wavelengths of 405 nm. Through the
overlap with the green fluorescence of tubulin, the PCD demonstrated a uniform distribution throughout the cytoplasm.
Moreover, photoluminescence intensity of the labeled cells
exhibited no obvious reduction in the experiment of continuous excitation for 30 min, which indicated that the PCD had a
remarkably high photostability with low photobleaching.
The biocompatibility of PCD was investigated using a CCK8 assay in MCF-7 cells and 293T cells (Fig. 3a and Fig. S11). The
PCD was much less cytotoxic than bPEI in both of the two
kinds of cells with the same method. Although cytotoxicity

Fig. 3 (a) Cell cytotoxicity of the PCD and bPEI (MCF-7 cells). (b) Agarose electrophoresis assay of DNA/PCD complexes at
different weight ratios. (c) Fluorescent images of EGFP expression obtained by an confocal laser scanning microscope in 293T
cells transfected with DNA/PCD complexes at weight ratio of 5. Incubation time: 48 h; scale bar is 50 lm. Green: green
fluorescent protein; blue: PCD; grey: bright field.

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of the PCD increased slightly with increasing concentration,

the concentration of PCD used in the in vitro evaluations were
significantly higher than that required for application such as
optical imaging of living cells.
To address whether PCD could be used as the gene carrier, its ability to condense with DNA was firstly evaluated
by agarose electrophoresis assay. The PCD was mixed with
DNA with different weight ratios (DNA/PCD) to form DNA/
PCD complexes. As can be seen from Fig. 3b, with the decrease of weight ratio, the PCD showed increased fluorescence while the intensity of DNA migration band
decreased significantly and finally vanished at or below
the weight ratios of 100, 50, 20, 10 and 5. To perform the
optimal transfection efficiency of PCD, the weight ratio of
five was selected for the further experiment. To visualize
the gene expression in vitro, transfection experiment in
293T cells were performed by using EGFP (enhanced green
fluorescent protein) as the reporter gene. As can be seen
from Fig. 3c, strong green fluorescence could be observed
from the 293T cell, which indicated the successful delivery
and expression of EGFP. The overlay images of the bright
field, the green fluorescence protein and blue PCD indicated
that the fluorescence of PCD could be used as the efficiency
label for the gene delivery. However, some of the blue PCD
could not superimpose with the green fluorescence protein,
which indicated that not all of the PCD contained intact
bPEI chains and the broken chains on the surface of PCD
lost their ability as gene carrier. Nevertheless, the high
transfection efficiency and bright blue fluorescence evidenced that PCD could be excellent carrier for both of cell
imaging and gene delivery.

4.

Conclusions

In conclusion, we have developed a novel method to prepare

multifunctional photoluminescent carbon using the oxidized
bPEI as starting material. The quantum yield of the PCD was
calculated to be 54.3%, which is one of the high values among
the CD reported to date. A significant advantage of the PCD
lies in its partially unchanged bPEI chain around the surface
of PCD, which demonstrated the potential for gene delivery.
The PCD exhibited extremely good biocompatibility. The
transfection experiment showed that the PCD could mediate
and visualize the gene transfection process in cells. The asprepared multifunctional PCD showed potential for applications in bioimaging and gene delivery.

Acknowledgements
This work was supported in part by the National Basic Research Program of China (2009CB930200) and National Natural
Science Foundation of China (81272453).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carbon.201
3.10.023.

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