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A mitochondria-targeted fluorescent probe based
on TPP-conjugated carbon dots for both one- and
two-photon fluorescence cell imaging†
Beibei Wang,‡
ab
Yanfang Wang,‡
ab
Hao Wu,
ab
Xiaojie Song,
ab
Xin Guo,
a
Demeng Zhang,
ab
Xiaojun Ma*
a
and Mingqian Tan*
a
A simple approach of a mitochondria-targeted fluorescent probe has
been demonstrated for the preparation of highly water-soluble and
fluorescent TPP-conjugated carbon dots (TPP-CDs) derived from low-
cost citric acid and urea. We found that the TPP-CDs are suitable for
both one- and two-photon mitochondria-selective imaging in living
cells.
Carbon dots (CDs), which have attracted a signicant amount of
attention, are found to be photochemically stable and emission
tunable with non-blinking uorescence and to have excellent
water solubility, and they can be produced cost-effectively on a
large scale.
1–4
Particularly, CDs without heavy metal content are
more environmentally friendly.
5–7
They have been shown to be
promising two-photon uorescent probes for biomedical
imaging and biosensing because of the low background signal,
deep tissue penetration, reduced photobleaching and low
phototoxicity associated with the use of near infrared excita-
tion.
8,9
Moreover, the existence of different functional groups,
such as –OH, –NH
2
and –COOH on the surface of CDs, makes it
possible to conjugate additional groups for generating new
multifunctional imaging probes, such as subcellular targeting
probes.
5
Mitochondria are crucial to cell life as the “energy plants”,
where oxidative phosphorylation and lipid oxidation occur.
10
A
wide range of diseases, including Friedreich's ataxia, Parkin-
son's disease, diabetes, and Huntington's disease, are caused by
damages to mitochondria.
10–12
Therefore, a specic visualiza-
tion of mitochondria is of great importance for their research
related to their structure and functions, as well as subcellular
theranostics.
12
The development of a mitochondria-targeted
uorescent probe with the ability of both one- and two-photon
emission is very interesting for the subcellular imaging of
mitochondria.
Herein, our strategy for the uorescent imaging of mito-
chondria in living cells is to create bifunctional probes that
contain both mitochondrial-targeting moiety and uorophore
derived from CDs. The nitrogen (N)-doped CDs exhibiting two-
photon emission ability were prepared to be the uorophore via
a facile strategy based on a one-step hydrothermal method. We
have selected the triphosphonium (TPP) head groups as the
targeting moiety to deliver the uorescent uorophore to
mitochondria, where these lipophilic cations can selectively
accumulate in the organelle due to the large membrane
potential gradient.
11
These design criteria can be met with the
approach outlined in Scheme 1 for the synthesis of N-doped
CDs and TPP-conjugated CDs (TPP-CDs).
The highly photoluminescent water-soluble CDs were
prepared by a cost-effective hydrothermal oxidation with citric
acid and urea as the carbon sources via one-pot synthesis.
Typically, the reactants were heated in a stainless steel autoclave
at 200 C for 10 h. By using various molar ratios of citric acid to
urea it was found that the molar ratios play a signicant role in
determining the nal quantum yield (QY) (Table S1†). The CDs
were found to possess free amine groups; this was veried by
color change through the reaction with ninhydrin (Fig. S1†).
The X-ray diffraction (XRD) analysis of the CDs powder
Scheme 1 Schematic illustration of the synthesis of N-doped TPP-
CDs for mitochondria-targeted cell imaging.
a
Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, 457 Zhongshan Road, Dalian 116023, China. E-mail: maxj@dicp.ac.cn;
mqtan@dicp.ac.cn; Fax: +86-411-84379139; Tel: +86-411-84379139
b
University of the Chinese Academy of Sciences, Beijing 100049, China
†Electronic supplementary information (ESI) available: Details of the synthesis,
characterization of CDs and TPP-CDS, two-photon mitochondria targeted
imaging. See DOI: 10.1039/c4ra07467j
‡Beibei Wang and Yanfang Wang contributed equally to this work.
Cite this: RSC Adv.,2014,4, 49960
Received 23rd July 2014
Accepted 19th September 2014
DOI: 10.1039/c4ra07467j
www.rsc.org/advances
49960 |RSC Adv.,2014,4, 49960–49963 This journal is © The Royal Society of Chemistry 2014
RSC Advances
COMMUNICATION
displayed one broad peak centered at 2q¼25(Fig. S2†), indi-
cating that the CDs are possibly amorphous materials attrib-
uted to highly disordered carbon atoms. The conjugation of
mitochondria targeting moiety, TPP, was realized via the ami-
dation of TPP-COOH to CDs-NH
2
using a modied EDC–NHS
strategy as described by Johnsson et al.
13
Aer purifying through
Sephadex G-25 gel column, the TPP-CDs were characterized by
transmission electronic microscopy (TEM). As shown in Fig. 1A,
the CDs are almost spherical nanoparticles with an average size
of 8.5 nm, which was conrmed by a detailed analysis of the
particle size for around 50 nanoparticles (Fig. S3A†). Aer
conjugation with TPP, the size of the TPP-CDs increased to
about 11 nm (inset of Fig. 1A) with a size distribution of 6–22
nm (Fig. S3B†). To conrm the size change in the CD particle
aer the conjugation of TPP, the gel permeation chromatog-
raphy analysis was performed. As shown in Fig. 1B, the TPP-CDs
display an elution time of 13.64 min, whereas the CDs have an
elution time of 13.70 min, suggesting the size increase of the
CDs aer the conjugation with TPP. Fourier transform infrared
(FT-IR) spectra (Fig. 1C) of TPP-CDs display a typical signal
decrease of N–H vibration at 1572 cm
1
and signal increase of
amide at 1673 cm
1
aer the amidation of TPP-COOH to CDs-
NH
2
as compared with CDs. The broad peak at 3400 cm
1
can
be attributed to the O–H vibration stretch of the carboxylic
moiety and N–H. The zeta potential of CDs is 18.0 mV due to
the existence of –OH/–COOH groups on the surface of CDs. Aer
conjugation with TPP, the zeta potential of TPP-CDs becomes
6.3 mV (Fig. 1D). Because of the presence of a large amount of
–COOH, –OH, and –NH
2
, the prepared TPP-CDs exhibit high
water solubility, which is essential for further bio-imaging
application.
UV-vis absorption spectra were used to investigate the
binding of TPP to CDs. As displayed in Fig. 2A, CDs show virtual
absorption peak at around 340 nm. Aer conjugation with TPP,
which has absorption peaks at 260, 267 and 275 nm, the UV-vis
spectrum of TPP-CDs contains the typical peaks of both CDs
and TPP, which suggests that TPP was successfully conjugated
onto the CDs. The CDs display an excitation-wavelength
dependent uorescence property (Fig. 2B), which was consis-
tent with the previous reports.
14,15
This phenomenon allows for
multicolor emission under different excitation wavelengths,
which is an important property for biomedical applications.
1
It
should be noted that the maximum emission peak of TPP-CDs
changed from 425 to 416 nm when it was excited at 340 nm
because of the introduction of the TPP moiety on the surface of
CDs (Fig. 2C). To conrm whether TPP-CDs are useful in
confocal imaging, the uorescence spectra were investigated as
displayed in Fig. 2D, which indicated that the TPP-CDs are still
able to emit uorescence at the excitation wavelength of
488 nm.
Using quinine sulfate as a standard, the uorescence QY of
TPP-CDs was about 15%. In addition, two-photon uorescence
emission from the CDs was observed upon excitation at 760 nm
with a femtosecond laser. To conrm whether the uorescence
originates from the two-photon absorption process with laser
excitation in the NIR, we examined the change of uorescence
intensity by varying the power of the 760 nm laser. As shown in
Fig. S4,†the photoluminescence (PL) intensity and the excited
laser power demonstrate an obvious quadratic relationship,
which reveals that the two-photon excitation is truly responsible
for the emission in nature. These results indicate that CDs and
TPP-CDs could be expected for cell imaging with two-photon
uorescence microscopy.
We also examined the effect of pH on the PL property of CDs
and TPP-CDs in Britton–Robinson buffer solution (Fig. S5†).
There are 22% and 17% increases of PL intensity for CDs and
TPP-CDs, respectively, when the pH values were changed from
2.0 to 12.0. The presence of many –NH
2
and –COOH groups on
the surface of CDs may account for the properties, which is
Fig. 1 TEM image of CDs (A). Inset shows the high-resolution TEM
image of TPP-CDs, scale bar ¼10 nm. Elution time curves of CDs and
TPP-CDs by using gel permeation chromatography (B). FT-IR spectra
(C) and zeta potential (D) of TPP-CDs, CDs and TPP. Zeta potentials of
CDs and TPP-CDs were recorded at a concentration of 10 mg mL
1
in
aqueous solution.
Fig. 2 UV-vis absorption spectra of TPP-CDs, TPP and CDs (A).
Fluorescence spectra of CDs (B) and TPP-CDs (C and D) at various
excitation wavelengths. Inset shows the photographs of the CD
aqueous solution observed under white light and UV light.
This journal is © The Royal Society of Chemistry 2014 RSC Adv.,2014,4, 49960–49963 | 49961
Communication RSC Advances
quite similar to amino acids. Amino acids are ampholytes and
have varying isoelectric points and dissociation constants.
16
Moreover, the emission wavelength showed a blue-shifor the
PL spectra for both CDs and TPP-CDs (Fig. S6†). The pH effect
indicates that the uorescent intensity would not be signi-
cantly quenched in acidic or alkaline media. In addition, both
CDs and TPP-CDs exhibit good photostability as compared with
uorescein (Fig. S7†). The uorescence intensity of both CDs
and TPP-CDs decreased about 10% aer being irradiated for
110 seconds, whereas the emission intensity of uorescein
decreased 55% during the same period. The CDs and TPP-CDs
derived from citric acid and urea with good photostability might
have potential applications in a wide range of pH in various
elds of nano-biotechnology.
The applicability for one- and two-photon bioimaging was
assessed by incubating HeLa cells with the CDs and TPP-CDs,
respectively. We observed that the HeLa cells became bright
under excitation at 405 nm, 488 nm (one-photon) and 760 nm
(two-photon) laser respectively (Fig. S8†). The cell images clearly
showed a high contrast uorescence of the CDs and TPP-CDs
around each nucleus, indicating that these CDs can be used
for living cells imaging without invading the nucleus in both the
one- and two-photon models.
17
The location of CDs and TPP-
CDs in live cells for both one- and two-photon imaging effi-
ciently matches. It should be noted that a low laser power of
0.27 W was sufficient to induce strong two-photon uorescence
of these CDs internalized in HeLa cells. The quantitative anal-
ysis with calibration curve shows that the uptake of TPP-CDs of
each cell is around 7.17 10
7
mg (Fig. S9†). The efficient
cellular uptake and strong one- and two-photon uorescence
emitting ability revealed that these carbon nanomaterials can
be used as potential probes for high contrast bioimaging.
The mitochondria-targeting properties of the TPP-CDs were
investigated by incubating the probes with HeLa cells using
non-targeted CDs as control. The confocal microscopy analysis
of the cells treated with TPP-CDs showed a greater uptake of
TPP-CDs than of CDs in the mitochondria of the cells (Fig. 3).
The details of the experimental procedure are described in the
ESI.†Quantitative analysis using the Olympus FluoView
“colocalization analysis”plug-in revealed the signicant coloc-
alization of the TPP-CDs with MitoTracker Deep Red in the
mitochondria of the cells (Pearson's correlation coefficient,
r¼0.62). In regards to the non-targeted CDs, a lower rvalue
(r¼0.46) was obtained as demonstrated by the different posi-
tion of the blue signals of CDs and the mitochondrial red
staining (Fig. 3). An analogous result in TCA-8113 tongue
squamous carcinoma cells treated with TPP-CDs was also
obtained as shown in Fig. S10.†The signicant colocalization of
the TPP-CDs with MitoTracker in the mitochondria of TCA-8113
was also found (r¼0.55, CDs control r¼0.19). The highly
efficient mitochondrial uptake of the targeted TPP-CDs can be
attributed to their high buffering capacity provided by lipophilic
TPP.
11
The delocalized positive charge of these cations enables
them to easily permeate lipid bilayers and to accumulate within
the mitochondria because of the large membrane potential
(150 to 70 mV, negative inside).
11
The plasma membrane
potential (30 to 60 mV, negative inside) also drives the
accumulation of these molecules from the extracellular uid
into isolated cells, from where they are concentrated further
within mitochondria.
11
The cellular uptake and intracellular trafficking of nano-
particles usually occurs along with competing pathways.
Colocalization with the lysosomes of both TPP-CDs and CDs
was also investigated; TPP-CDs resulted in a lower accumula-
tion (r¼0.17), whereas CDs showed greater uptake (r¼0.33) in
the lysosomes of the HeLa cells (Fig. 4). The data show that the
uptake of targeted TPP-CDs by mitochondria is considerably
higher than that by lysosomes during the trafficking process,
suggesting the good mitochondria accumulation ability of TPP-
CDs. All these results reveal that TPP-CDs target the cellular
mitochondria in living samples.
The biocompatibility of probes is one of the most important
properties to consider their bio-applications. MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-phenyltetrazolium bromide) assay in
HeLa cells was carried out to investigate the cytotoxicity of the
as-prepared CDs and TPP-CDs. As shown in Fig. S11,†cell
viability remains more than 84% aer incubation with a rela-
tively high concentration of TPP-CDs (6 mg mL
1
) for 8 hours,
demonstrating the lower toxicity of the NPs to living cells. In
addition, the viability of HeLa cells incubated with CDs was
more than 96%. It is clear that the cytotoxicity of both CDs and
TPP-CDs was relatively low at shorter incubation time, which is
essential for their further applications in living cells imaging.
In summary, we have developed a general and facile method
to prepare a mitochondria-targeted uorescent probe based on
Fig. 3 Subcellular localization of mitochondria targeted TPP-CDs and
non-targeted CDs after 4 h incubation with HeLa cells and colocali-
zation with MitoTracker Deep Red. Scale bar ¼20 mm. Excitation
wavelength for MitoTracker is 635 nm.
Fig. 4 Subcellular colocalization of TPP-CDs and non-targeted CDs
with LysoTracker after 4 h incubation with HeLa cells. Scale bar ¼20
mm. Excitation wavelength for LysoTracker is 543 nm.
49962 |RSC Adv.,2014,4, 49960–49963 This journal is © The Royal Society of Chemistry 2014
RSC Advances Communication
TPP modied uorescent CDs derived from low-cost citric acid
and urea. The resulting TPP-CDs were well-dispersed in water
and possess excitation tunable photoluminescence, high pho-
tostability, low cytotoxicity and suitability for their use in
various pH conditions. Signicantly, the TPP-CDs showed clear
two-photon emission under the excitation of NIR uorescence
with the possibility for two-photon imaging of live cells. More-
over, the TPP-CDs were applied for mitochondria-targeted
tumor cell imaging, which were capable of producing
outstanding mitochondria accumulation. All these results
demonstrated that the prepared TPP-CDs may have potential to
be a uorescent probes for biomedical applications.
Acknowledgements
This work was supported by the National Nature Science
Foundation of China (91227126), National Special Fund for Key
Scientic Instrument and Equipment Development
(2013YQ17046307) and the Nature Science Foundation of
Liaoning Province, China (2013020177).
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