Materials Today  Volume 19, Number 7  September 2016 RESEARCH

RESEARCH: Review

Carbon dots: large-scale synthesis,

sensing and bioimaging
Jia Zhang and Shu-Hong Yu*
Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemistry, Collaborative Innovation Center
of Suzhou Nano Science and Technology, Hefei Science Center, CAS, CAS Center for Excellence in Nanoscience, University of Science and Technology of China,
Hefei, Anhui 230026, PR China

Emerging as a potent alternative to classical metal-based semiconductor quantum dots (Qdots), carbon
dots (Cdots) possess the distinctive advantages of convenient synthesis, prominent biocompatibility,
colorful photoluminescence, and low cost. After almost a decade of extensive studies since their
discovery, Cdots have widely been applied in bioimaging, sensing, catalysis, optoelectronics, energy
conversion, etc. In this review, we first highlight the synthetic methods for Cdots in a macroscale
manner. Second, we briefly discuss the fundamental mechanisms underlying the photoluminescence
(PL). Third, we focus on their applications in sensing and bioimaging (including imaging-guided
therapy). Some thoughts on future developments of Cdots are demonstrated as concluding remarks.

Introduction sensing, bioimaging, nanomedicine, catalysis, optoelectronics,

Carbon dots (Cdots) have been acknowledged as discrete, quasi-
spherical particles with sizes below 10 nm [1]. They generally
possess a sp2 conjugated core and contain suitable oxygen content
in the forms of multiple oxygen-containing species represented by
carboxyl, hydroxyl, and aldehyde groups. In 2004 during the
electrophoretic fractionation of arc-discharge soot, fluorescent
carbon nanoparticles with vertical sizes at about 1 nm were isolat-
ed with the assertion from the discoverers that ‘‘they promise to be
interesting nanomaterials in their own right’’ [2]. However, it was
until two years later that the research work from Sun’s group
ignited a burst of interests in these ‘‘interesting carbon nanoma-
terials’’ which were coined afterwards as carbon dots, or carbon
nanodots, or carbogenic quantum dots [3]. Photoluminescence
(PL) is the predominant property of Cdots with which researchers
are mostly interested. Excitation wavelength-dependent [3–7] and
excitation wavelength-independent [8–10] PL behaviors were both
demonstrated. Ever since 2006 the number of scientific publica-
tions on Cdots increases exponentially, and such explosion of
interests is primarily due to the several merits of Cdots, including
simple synthesis, low cost, and excellent biocompatibility. Until
now, the applications of Cdots have been widely demonstrated in
*Corresponding author. Yu, S.-H. (shyu@ustc.edu.cn)
and energy conversion/storage. More than half of these are estab-
lished upon their PL properties. In fact, the colorful photolumi-
nescence, high photostability, and low toxicity of Cdots enable
them strong competitors and potential alternatives to those heavy
metal-based semiconductor quantum dots (Qdots) currently in
use.
Even though a number of reviews on the syntheses, properties,
and applications of Cdots were published [1,11–13], a more focus
overview on large-scale synthesis of Cdots for sensing and bioima-
ging is still missing. In this review, we start to emphasize those
methods compatible with large-scale synthesis. Then we discuss
the origins of their PL corresponding to Cdots by different syn-
thetic methods. Next we provide a representative account of their
applications in sensing, bioimaging, and imaging-guided therapy.
As concluding remarks, some perspectives on the future develop-
ments of Cdots are to be highlighted.
Large-scale synthesis of carbon dots
The development of large-scale synthetic methods for Cdots is
fundamental to their applications. Basically most of the current
synthetic methods can be used to produce carbon dots in large
mass regardless of the intrinsic nature of raw materials, while in
practice, only a few works carried out the mass production. With

1369-7021/ß 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/
382 j.mattod.2015.11.008
Materials Today  Volume 19, Number 7  September 2016
the Chinese ink as raw material, Yang et al. [14] obtained 120 g of
Cdots in one pot of synthesis (Fig. 1a). Even though carbon
materials are ideally suitable precursors for the macroscale synthe-
sis of carbon dots, using a large amount of strong acids might cause
severe pollution, hence arousing great environmental concerns.
From this consideration, biomass and molecules seem to be better
alternatives to carbon materials. Owing to a large number of
biomass both in diversity and amount, the possibility of large-
scale synthesis of Cdots from biomass is remarkably great. Sahu
et al. [15] mixed orange juice with ethanol to prepare 0.4 g of Cdots
by hydrothermal treatment at 120 8C for 2.5 h (Fig. 1b). Although
the yield still needs to be improved, it is reasonable to believe using
liquid biomass is an appropriate strategy to produce carbon dots in
large mass if technologically viable. Compared to using liquid
biomass, the use of solid biomass may be a better solution to scale
up the synthesis due to their much longer shelf life and more
convenient scalability in mass of precursors. Chen and coworkers
[16] used chicken eggs to prepare Cdots by plasma-induced pyrol-
ysis. For printing applications, up to 10 g of these Cdots was
prepared. Chang’s group [17] employed coffee grounds as raw
material and they could obtain 120  30 mg of Cdots from 1 g
of coffee grounds. Most recently, we [18] proposed to scale up the
synthesis of Cdots by adopting bee pollens as carbon source, with
more than 3 g of Cdots produced from 10 g of bee pollens (Fig. 1c).
Instead of using apparently sanitary biosources, Park et al. [19,20]
used food wastes and harmful cyanobacteria to synthesize Cdots in
large scale.
During the enlarged syntheses, reproducibility is critically im-
portant for the potentially technological applications of carbon
dots, while most of preparation methods did not mention it. We
first investigated the reproducibility regarding the synthesis of
Cdots [18]. Taking rapeseed bee pollen as an example, when the
FIGURE 1
Diagrams for the large-scale synthesis of carbon dots by adopting (a) Chinese ink (Reprinted with permission from Ref. [14]. Copyright 2014 Royal Society of
Chemistry). (b) orange juice (Reprinted with permission from Ref. [15]. Copyright 2012 Royal Society of Chemistry). (c) bee pollens, and (d) sucrose as the
carbon source (Reprinted with permission from Ref. [21]. Copyright 2013 Royal Society of Chemistry).
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mass concentration of bee pollen was set constant, we found that
the reaction volumes would not vary the production yield of Cdots
significantly. With respect to feeding mass of 0.5, 1, 2, and 10 g of
bee pollen, after 24 h of hydrothermal treatment, the average yield
weighted 30.2  1.7%. And for five times of repeated syntheses at
feeding mass of 1 g, the yields were averaged to be 30.8  1.1%.
The fluorescence quantum yields of these Cdots were 9.5  0.74%.
Moreover, the scale-up synthesis did not affect the particle size
notably.
Complementary to the use of biomass, using molecular pre-
RESEARCH: Review
cursors is a straightforward strategy to produce carbon dots with
relatively high yields. Chen et al. [21] developed a simple step to
synthesize Cdots at gram scale with a yield of 41.8% by carboni-
zation of sucrose in oleic acid. In their method, 20 g of sucrose was
heated in oleic acid at 215 8C for 5 min under vigorous stirring and
N2 protection. After purification, 8.36 g of product was obtained
(Fig. 1d). Bhunia et al. [7] proposed a controlled carbonization
approach of carbohydrate to generate Cdots in gram scale. Car-
bonization was conducted in different solvents with different
dehydrating agents and at temperatures from 80 to 300 8C. Until
now the large-scale synthesis of carbon dots by those reported
methods only leads to yields in gram scale; therefore, it is impera-
tive and challenging to further enlarge the synthesis. In the
meantime, the reproducibility and environmental issue should
be considered properly.
Photoluminescence of carbon dots
Wide variations exist in the literature regarding the PL properties
of Cdots, such as the dependence of emission on excitation
wavelength (lex) or not, or the dependence of quantum yield
on surface modification. Sun’s group [3] found that the acid-
treated nanoscale carbon particles did not exhibit noticeable PL
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until after the surface passivation by amine derivative polymers.
The requirement for surface passivation to become photolumines-
cent suggested mechanistically that the PL from Cdots might be
attributed to surface energy traps that became emissive upon
stabilization as a consequence of surface passivation. The radiative
recombination of excitons was regarded as the mechanism for the
luminescence emissions. Using graphite rods as the electrodes and
NaOH/ethanol as electrolyte, Kang and coworkers [22] synthesized
Cdots and fractionated them into samples of varying sizes. Along
with the increase of size, the Cdots emitted blue, green, yellow,
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and orange, respectively, under identical lex. Treated by H2 plasma
to remove surface oxygen species, there was no appreciable change
in the PL spectra. Thus they attributed the luminescence emissions
of Cdots to the quantum-sized graphite structure rather than the
carbon-oxygen surface. Bao et al. [23] prepared luminescent Cdots
of different sizes with narrow size distribution by electrochemical
tuning of carbon fibers at different potential. At same lex, the
emission peak shifted to a lower energy position as the sizes of
Cdots decreased. This is different from the quantum-confinement
effect aforementioned [22]. The smaller Cdots formed at a higher
potential were more oxidized, and thus bared more oxygen-related
surface states. Therefore, they speculated the red shift of emission
of Cdots due to variation in surface states rather than a size effect.
In addition to electrochemical treatment, chemical process can
also alter the PL of carbon dots in terms of quantum yield and
maximum emission. Zheng et al. [24] prepared Cdots emitting
green luminescence by the oxidation of graphite oxide with nitric
acid. The dots emitted in blue after reduction by NaBH4 with
maximum emission wavelength shifting from 520 to 450 nm.
Moreover, an apparent increase of quantum yield from 2% to
24% was induced by the chemical reduction. The emission spectra
of the reduced Cdots remained constant by excitation at from 260
to 360 nm, but shifted to lower energies at lex from 380 to 460 nm.
In comparison, the unprocessed Cdots showed a lex-independent
emission profile, similar with the luminescent behavior of carbon
dots by electrochemical oxidation of graphite [8]. This suggests
that the intrinsic luminescence from graphitic core networks may
be responsible for the emission at short wavelengths, but other
deactivation pathways dominate the luminescence at longer
wavelengths [25]. The bright luminescence of Cdots synthesized
by Pan et al. from the pyrolysis of EDTA sodium salt not only
depended on lex, but also on pH and solvent [26]. After excluding
the possibilities of carboxyl group and large graphene clusters for
the luminescent emission, they proposed small sp2 clusters of
graphene embedded in carbon dots to be the rightful cause.
One problem pertaining to their conjecture is the lack of consid-
eration of influence from the carbon backbone. By means of
ultrafast spectroscopy, Sun and colleagues [27] revealed that the
hybridization of the surface groups with the carbon backbone
formed the emissive centers existing in those green luminescent
carbon dots.
In above studies, most of the Cdots are produced from carbon
sources, while many other luminescent Cdots are reported being
derived from the thermal condensation between molecules. Gian-
nelis and coworkers [28] developed a new strategy to explain the
PL mechanism of Cdots by temperature-controlled condensation
and carbonization. Pyrolysis of citric acid and ethanolamine at low
temperature (180 8C) produced a molecular fluorophore with a
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Materials Today  Volume 19, Number 7  September 2016
remarkably high quantum yield of 50%. A behavior of single,
excitation-independent PL was observed. At higher temperature
(230 8C), pyrolysis led to particles of spherical symmetry with an
average diameter of 19 nm. The emission quantum yield decreased
to 15%, indicating a large amount of the organic fluorophores was
used as building blocks in favor of the carbogenic domains.
Besides, excitation with longer wavelengths (above 400 nm)
started to induce the red shift in the emission position, a generic
feature of carbon dots. Further pyrolysis at higher temperatures
(300 and 400 8C) resulted in Cdots with much minimized quan-
tum yields since most of amide-containing fluorophores were
consumed and the PL arose mostly or exclusively from carbogenic
cores. Thus this study shows that for carbon dots prepared from
molecules, the dependence of emission on excitation is due to the
formation of carbogenic cores and the excitation-independent
emission to organic fluorophores.
After years of intensive researches, the exact mechanism for the
PL of carbon dots is still debatable. A reasonable explanation is that
different PL mechanisms may be involved for carbon dots by
different synthetic methods. Even so, it does not impede their
applications in broad areas. In the following section, we will focus
on the use of carbon dots in sensing and bioimaging.
Carbon dots in sensing and bioimaging
Sensing of metal ions
A number of carbon dots have been developed as direct fluorescent
sensors for metal ions. Dong et al. [29] synthesized highly lumi-
nescent Cdots with quantum yield of 42.5% using citric acid and
branched poly(ethylenimine) (bPEI) as the precursors by multistep
thermal pyrolysis. The plentiful amine groups at the surface of
Cdots captured Cu2+ ions through metal coordination to form a
complex whose absorption band overlapped partially with the
excitation and emission spectra of Cdots. In this way, the fluores-
cence of solution was quenched because of the possible inner filter
effect (IFE). On the basis of the upconversion fluorescence of PEI-
capped Cdots, Salinas-Castillo et al. [30] developed a sensor of Cu2+
ion with improved selectivity. Instead of utilizing one signal,
adopting a reference signal can provide build-in correction to
avoid external effects. With such strategy, Tian and coworkers
[31] developed a dual-emission nanohybrid system consisting of
Cdots and CdSe/ZnS Qdots for ratiometric fluorescent detection of
Cu2+ ion in vitro and in cells. As shown in Fig. 2a, prior to the
addition of Cu2+, the emission at 485 nm due to the presence of
Cdots is more intense than that at 644 nm due to the presence of
Qdots, and thus a blue fluorescence is observed from the solution
under excitation. After the addition of Cu2+, the molecular recep-
tors on the Cdots surface bind with Cu2+ ions specifically to
quench the fluorescence of Cdots. Since the emission at 644 nm
remained constant, the decrease of the blue fluorescence results in
an observable fluorescent color change to pink. Even though there
is little change in the detection performance spectroscopically, the
involvement of the second signal as reference does improve the
sensing sensitivity visually (Fig. 2b). As an extension of the fluo-
rescent sensing platform, the same group immobilized the molec-
ular receptor-functionalized Cdots on the electrode, developing a
highly selective electrochemical strategy for the determination of
Cu2+ ion in complex brain system with a detection limit of 100 nM
[32]. Different from the fluorescence ‘‘on-off’’ paradigm, Chang’s
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RESEARCH: Review
FIGURE 2
(a) Diagram for the dual-emission fluorescent sensing of Cu2+ ion based on a CdSe/ZnS-Cdots nanohybrid. (b) Comparison between fluorescent spectra and
fluorescent colors of the nanohybrid probe and Cdots probe after exposure to CuCl2. The concentrations of CuCl2 from 1 to 6 for the spectra and from left
to right for the colored solutions both correspond to 0, 10, 20, 30, 40, and 50 mM, respectively. Reprinted with permission from Ref. [31]. Copyright 2012
John Wiley & Sons. (c) Schematic illustration of graphene oxide-based sensor system for the detection of Hg2+ ion using Cdots-labeled ODN as the
recognition element and signal reporter. Reprinted with permission from Ref. [36]. Copyright 2015 Elsevier. (d) Sensing principle of the N-doped Cdots based
probe for Fe3+. Reprinted with permission from Ref. [37]. Copyright 2014 American Chemical Society.

group [33] proposed a method to detect Cu2+ ion by the fluores-
cence ‘‘off-on’’ strategy with o-phenylenediamine (OPD)-derived
Cdots.
Today the widespread pollution of mercuric (Hg2+) ion raises
serious environmental and health concerns. By the use of carbon
dots, some sensors of Hg2+ ion were developed. Utilizing pomelo
peel as the carbon source, Lu et al. [34] prepared Cdots for the rapid
assay of Hg2+ ion with agreeable sensitivity and selectivity. Zhang
and coworkers [35] designed a specific fluorescence ‘‘turn-on’’
nanosensor for Hg2+ ion. A bis(dithiocarbamato)copper(II) com-
plex (CuDTC2) was covalently attached onto the Cdots surface
causing the fluorescence quenching by a combination of electron
transfer due to Cu2+ and energy transfer due to the complex. Hg2+
could displace Cu2+ in the complex efficiently and thus shut down
the pathways of electron transfer and energy transfer, thereby
turning the fluorescence on. The work by Cui et al. [36] demon-
strates that the sensing paradigm previously proposed to detect
Hg2+ using other optical probes can be also in function at the use of
carbon dots (Fig. 2c). They first modified Cdots with thymine (T)-
rich oligodeoxyribonucleotide (ODN). Graphene oxide could
quench the fluorescence of the modified Cdots via fluorescence
resonance energy transfer (FRET) owing to the preferential adsorp-
tion of ODN on its surface. In the presence of Hg2+, a T-Hg2+-T
duplex was formed, and the fluorescence recovered due to desorp-
tion of ODN.
Fe3+ ion is also commonly fluorescently detected by Cdots.
Zhang et al. [37] used a solid-phase synthesis approach to generate
blue luminescent Cdots with a high quantum yield of 31%. They
suggested that the strong coordination of Fe3+ ion to oxygen-rich
groups on Cdots surface induced the fluorescence quenching by
nonradiative electron transfer (Fig. 2d), offering a fluorescence
‘‘turn-off’’ avenue for the determination of Fe3+ ion. Similar trend
of changes in fluorescence were also observed for other label-free
Cdots-based probes for the detection of Fe3+ ion [38,39].
Several other metal ions, including K+ [40], Cr6+ [41], Pb2+ [42],
Zn2+ [43] and Ag+ [44], were also detected with Cdots by fluores-
cence. Moreover, Cdots-improved chemiluminescence (CL) per-
formance was employed to fabricate a sensor of Co2+ ion [45].
Albeit the larger number of sensors for metal ions based on carbon
dots, a lack of sufficient selectivity is often encountered, making
them susceptible to interference from other ions or substances.
Therefore, labeling carbon dots with suitable molecular receptors
with ion specificity should primarily be considered in future
studies.
Sensing of anions
Lin et al. [46] first discovered the CL response of PEG-capped carbon
dots induced by peroxynitrous acid. The CL intensity increased in
proportion with the concentration of nitrite in the range from 0.1 to
10 mM, with a detection limit of 53 nM. They conferred the CL
behavior to the radiative recombination of hole-injected and elec-
tron-injected Cdots (Fig. 3a). Huang’s team [47] reported a visual
method to detect phosphate by a probe of Eu3+-Cdots with fluores-
cence ‘‘off-on’’ response (Fig. 3b). In the presence of Eu3+ ion, the

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FIGURE 3
(a) Schematic illustration of the CL mechanism of Cdots-NaNO2-H2O2 system. Reprinted with permission from Ref. [46]. Copyright 2011 American Chemical
Society. (b) Diagram for sensing of phosphate based on the modified Cdots mediated by Eu3+ ion. Reprinted with permission from Ref. [47]. Copyright 2011
Royal Society of Chemistry. (c) Graphic illustration of the sensing principle based on the inner filter effect of metal nanoparticles for the detection of cyanide
ion with carbon dots as the fluorescent probe. Reprinted with permission from Ref. [51]. Copyright 2015 Springer.
aminoundecanoic acid-passivated Cdots aggregated through the
coordination of Eu3+ ion with the carboxylate groups, leading to
the decrease of fluorescence. At the subsequent addition of phos-
phate, it reversed the Cdots to disperse due to a higher affinity of
Eu3+ to phosphate. As a result, the optical signal increased backward.
By such avenue, they achieved a linear detection range of phosphate
between 0.4 and 15 mM with a detection limit of 51 nM. This
strategy of sensing anions mediated by a metal ion has also been
applied to detect iodide [48]. Using sweet pepper as the carbon
source, Yin et al. [49] synthesized Cdots that showed down- and up-
conversion fluorescent properties toward the detection of hypo-
chlorite. Tian and coworkers [50] fabricated a Cdots-based ratio-
metric fluorescent biosensor for superoxide anion (O2). The
hybrid probe emitted green fluorescence initially, and in the pres-
ence of the target, the response signal observed at 610 nm due to the
recognition element increased to a more extent than the signal at
525 nm resulting from carbon dots, inducing the fluorescence
change to red. With the use of noble metal nanoparticles to quench
the fluorescence of Cdots by IFE, we developed a sensor for cyanide
ion with fluorescence ‘‘turn-on’’ (Fig. 3c) [51]. The Cdots prepared
from bee pollen emitted weakly in the presence of gold or silver
nanoparticles due to their absorption of excitation and emission
light. After the addition of cyanide ion, the particles were etched,
leading to decrease in absorbance and restoring the IFE-minimized
emission. This study broadens the applications of label-free carbon
dots in sensing of anions.
Sensing of molecules
Photoluminescence is still the preferentially employed property of
Cdots for the detection of molecules. Wang et al. [52] prepared
Cdots-silica aerogel hybrid material for the sensing of NO2. Dif-
ferent from the monotonic change of fluorescence, Yan et al. [53]
implanted a second signal to make a ratiometric fluorescent probe
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Materials Today  Volume 19, Number 7  September 2016
for the sensitive detection of NO2. The blue fluorescence of Cdots
was insensitive to NO2, serving as a reference signal, and the red
florescence of CdTe Qdots was efficiently quenched by NO2, which
resulted in a visible color change from orange red to blue upon
exposure to the target (Fig. 4a). The selectivity test result showed
that the sensor was highly specific to NO2 among a series of other
species, such as H2O2, NO, HClO, CO2, CO, etc.
The other type of ratiometric sensing based on carbon dots is
established by the surface modification of analytes-specific organic
molecules to make the FRET process happen. Adopting this strategy,
Wu and colleagues [54] reported a fluorescent sensor for H2S. The
Cdots functioned as the energy donor, and the naphthalimide azide
turned to the energy acceptor in the presence of H2S. As a conse-
quence, with the gradual increase of H2S concentration, the blue
fluorescence of Cdots minimized, and the green fluorescence result-
ing from the naphthalimide azide product increased, thereby in-
ducing a color change of blue to green distinguished by naked eye
(Fig. 4b). By a change of the recognition element to OPD-containing
naphthalimide, they also developed a ratiometric sensor for detect-
ing NO [55], and by the covalent linking of an additional mitochon-
dria-targeting ligand, they further built up a FRET-based nanoprobe
for imaging mitochondrial H2O2 in living cells [56].
By the use of an interactive mediator, a few examples of fluo-
rescent sensors for molecules based on label-free Cdots have been
reported. Qian et al. [57] presented a fluorescent ‘‘off-on’’ ap-
proach to monitor alkaline phosphatase activity on the basis of
aggregation and disaggregation of Cdots by the mediation of
cerium ion. Li et al. [58] demonstrated a nanoprobe consisting
of hexagonal cobalt oxyhydroxide (CoOOH) nanoflakes and Tris-
derived Cdots for fluorescent ‘‘turn-on’’ assay of ascorbic acid (AA)
in rat brain. Cdots provided the signal readout, and the CoOOH
served as the recognition element for AA (Fig. 4c). After hybrid-
ization, the fluorescence of Cdots was quenched by CoOOH due to
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FIGURE 4
Schematic representations of the sensing of (a) NO2 (Reprinted with permission from Ref. [53]. Copyright 2015 American Chemical Society). (b) H2S
(Reprinted with permission from Ref. [54]. Copyright 2013 Royal Society of Chemistry). (c) ascorbic acid (Reprinted with permission from Ref. [58]. Copyright
2015 American Chemical Society) and (d) thrombin (Reproduced with permission from Ref. [60]. Copyright 2012 Royal Society of Chemistry).

FRET. Upon the addition of AA, the enediol group of AA could
reduce CoOOH to Co2+ ion, leading to collapse of the hybridized
structure, releasing Cdots, and restoring the fluorescence signal.
Chen et al. [59] developed a nanoprobe based on terbium (Tb3+)
coupled carbon dots that was used to detect dipicolinic acid (DPA)
as an anthrax biomarker. In the presence of DPA molecule, the
emission at 440 nm of Cdots remained constant as a reference
signal, and the Tb3+ ions exhibited enhanced sharp emission
especially at 544 nm, realizing ratiometric detection of DPA by
fluorescence change from blue to green visibly.
By combining DNA with carbon dots, DNA-specific biomole-
cules can be sensed fluorescently. Qu and coworkers [60] designed
target-induced assembly of a sandwich nanostructure for Cdots-
based fluorescent detection of thrombin (Fig. 4d). Two thrombin
aptamers were individually modified on the surfaces of Cdots and
silica nanoparticles to produce two separate assembling units. In
the presence of thrombin, the smaller unit Cdots attached around
the surface of the bigger unit silica nanoparticles due to the specific
aptamer–thrombin interaction. After subject to centrifugation and
redispersion, the product of sandwich structure could be quanti-
tatively detected by fluorescence emission spectra or fluorescence
microscopy images, from which the amount of thrombin was
quantified.
Self-assembly of carbon dots leading to aggregates is often
accompanied by the fluorescence quenching, which provides
the basis for the development of biosensing systems. Using phe-
nylboronic acid as the sole precursor, Shen and Xia synthesized
boronic acid-functionalized Cdots for the determination of glu-
cose in blood samples [61]. Owing to the specific interaction
between boronic acid species and glucose, Cdots became severely
aggregated in the presence of glucose, and the fluorescence
switched off. Stevens and coworkers [62] integrated anionic Cdots

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FIGURE 5
Laser scanning confocal microscopy images of ethylenediamine-carbon dots labeled L929 cells. Reprinted with permission from Ref. [68]. Copyright 2012
Royal Society of Chemistry.
and oppositely charged biopolymers into hybrid materials by
electrostatic self-assembly for the design of label-free biosensors.
Depending upon the biopolymer, ionic self-assembly generated a
series of hierarchical structures that exhibited aggregation-in-
duced fluorescence quenching of the Cdots. Upon competitive
binding of glycosaminoglycans (GAGs) to biopolymers or enzy-
matic hydrolysis of the protein backbone, the hybrid structures
disassembled along with the recovery of fluorescence, realizing the
simple and sensitive detection of GAGs (heparin) and protease
(trypsin). Contrary to the aggregation-induced fluorescence
quenching, a phenomenon of aggregation-induced emission en-
hancement (AIEE) effect for Cdots was recently discovered and
used for sensing applications. Gao et al. [63] first found that Cdots
prepared from hydrothermal treatment of C60 molecules in a
mixture cetyltrimethylammonium bromide (CTAB), H2O2, and
NaOH displayed AIEE. The addition of adenosine triphosphate
(ATP) caused the Cdots aggregate and the fluorescence increase.
This abnormal AIEE effect of Cdots is assumed to attribute to the
reduced non-radiative decay arising from aggregation. Zhang et al.
[64] also found the similar AIEE effect for boron-doped Cdots in
the presence of glucose. They postulated that the binding of
glucose to boronic acid groups on the Cdots surfaces created
Cdots-glucose aggregates of rigid structure, thus restricting the
intramolecular rotations and boosting the fluorescence greatly.
Since the presence of two cis-diol units in glucose was pivotal to
bridging Cdots, high selectivity toward glucose was guaranteed
over its isomers such as fructose, galactose, and mannose.
In addition to the PL, other physical properties of Cdots were
also harnessed to fabricate sensors for biomolecules. Dai et al. [65]
reported a Cdots-based electrochemiluminescence (ECL) immu-
nosensor for a-fetoprotein. Huang et al. [66] built up an electro-
chemical platform based on Cdots and chitosan to develop a
biosensor for sensitive and selective determination of dopamine.
Moreover, Shi et al. [67] found Cdots from the oxidative treatment
of candle soot exhibited intrinsic peroxidase-like activity, based on
which a colorimetric sensor for glucose has been made.
Bioimaging
Carbon dots own distinctive advantages marked by multicolor
emission profile, small sizes, low cytotoxicity, prominent
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Materials Today  Volume 19, Number 7  September 2016
biocompatibility, and excellent photostability, enabling them as
an ideal candidate for fluorescence imaging. Zhai et al. [68] syn-
thesized photoluminescent Cdots with a quantum yield of 30.2%
to label L929 cells. As shown in Fig. 5, the Cdots-incubated cells
emitted blue, green, and red fluorescence upon excitation at 405,
488, and 543 nm, respectively. Those areas from which the fluo-
rescence was emitted reflected the presence of Cdots, which were
distinctly located in the cell membrane and cytoplasm, especially
around the cell nucleus. Until now, nearly all Cdots without
specific modifications basically label the cell membrane and cyto-
plasm areas without reaching the nuclei mainly through passive
endocytosis [20].
Sun’s group [69] first demonstrated the potential of carbon dots
in cell imaging with two-photon (TP) luminescence microscopy. It
showed the TP absorption cross-section of Cdots (passivated by an
imine polymer) was comparable to those of the best-performing
CdSe/ZnS core-shell Qdots. Tian’s group [70] proposed a Cdots-
based nanoprobe for TP fluorescent imaging of pH variation in
living cells and tissues. In order to response to the change of [H+]
selectively, a receptor molecule 40 -(aminomethylpheynl)-
2,20 :60 ,200 -terpyridine (AE-TPY) was covalently bound on the Cdots
surface. In the beginning, the probe emitted feeble fluorescence at
498 nm with TP excitation at 800 nm; while as the acidity de-
creased, the fluorescence signal became more intense. Since exci-
tation at 800 nm is most preferable for tissue penetration, they also
realized the TP imaging of pH gradients in tumor tissues at a depth
of 65–185 mm.
In contrast to unmodified Cdots, the specially functionalized
Cdots can label cells more efficiently and specifically. Song et al.
[71] prepared folic acid (FA)-modified Cdots by covalent linking of
FA to amine-capped carbon dots. Due to the specific interaction
between FA and folate receptor (FR) molecule, the Cdots could
distinguish cancer cells overexpressing folate receptor from nor-
mal cells. Lee et al. [72] also developed an aptamer-conjugated
Cdots imaging probe for targeting cancer cells. Similarly, Ruan
et al. [73] observed glioma imaging with higher sensitivity over
normal brain tissues by using PEGylated carbon dots after decora-
tion of angiopep-2, a ligand that could target glioma cells.
Sun’s group [74] first used Cdots for optical imaging in vivo by
adopting three injection avenues. Following this study, Kang and
Materials Today  Volume 19, Number 7  September 2016 RESEARCH

RESEARCH: Review
FIGURE 6
In vivo and ex vivo fluorescent imaging at 2 h after intravenous injection of carbon dots. (a) 3D reconstruction of in vivo imaging of PEG-Cdots treated mice.
(b) 3D reconstruction of in vivo imaging of An-PEG-Cdots treated mice. (c) Ex vivo imaging of glioma bearing brain. (d) Semiquantitative fluorescent intensity
of brain and glioma. *p < 0.05. (e) G/N ratio of PEG-CDs and An-PEG-Cdots group. Reprinted with permission from Ref. [73]. Copyright 2014 American
Chemical Society.

FIGURE 7
3D two-photon confocal fluorescence images (under 810 nm excitation) and corresponding bright-field images of Cdots-FA-DOX (3 mg ml1) conjugate-
incubated glomerular tissues, accumulated along the z-direction at a depth of 65–300 mm, incubated under pHs of (a) 7.4, (b) 6.5, and (c) 5.5. Different
imaging channels are displayed horizontally for each sample (from left to right): Cdots channel (475–525 nm), DOX channel (575–625 nm), bright field, and
overlay images. The scale bars in all images are 60 mm. Reprinted with permission from Ref. [78]. Copyright 2013 John Wiley & Sons.

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coworkers [75] made a thorough investigation to in vivo near
infrared (NIR) fluorescence imaging, biodistribution, and toxicol-
ogy of unpassivated carbon dots prepared from acid oxidation of
carbon nanotubes and graphite. After intravenous injection, the
Cdots accumulated heavily in the reticuloendothelial system (RES)
and in the kidney, and they were gradually excreted via renal and
fecal pathways. Results of a panel of biochemical markers indicat-
ed that no obvious toxic effects were observed for these Cdots in
mice. Afterwards, Ko et al. [76] tested the capability of Cdots for in
vivo NIR fluorescence imaging of tumor-bearing mice. Ruan et al.
RESEARCH: Review
imaging-guided PDT was investigated in nude mice. After intrave-
nous injection, the fluorescence from Ce6 became visible in tumor
sites at 2–4 h post-injection and reached a plateau at 4–8 h post-
injection, and thus the suitable time point to implement PDT was
selected at 8 h. By the substitution of Ce6 with zinc phthalocyanine
and the introduction of FA as a targeting agent, Choi et al. [80]
developed a similar Cdots-based nanoprobe for targeted PDT and
fluorescence bioimaging in vitro and in vivo.
To date, the known PSs possess high SO quantum yields but
absorb in the visible spectra. Therefore, from the clinic perspective,

[73] observed enhanced targeting efficiency of PEG-Cdots to glio- new PS systems that can extend the use of PDT to deeper-seated
ma through the modification of angiopep-2 in cells experiment, tumors with light falling within the phototherapeutic window
and they further performed in vivo study to demonstrate the (780–950 nm) shall mostly be desired. In view of the excellent TP
advantages of the modified probe (An-PEG-Cdots), shown in absorption characteristics and biocompatibility of carbon dots,
Fig. 6. At 2 h after intravenous injection of both carbon dots into their hybridization with PS molecules shall produce ideal PDT
mice, in vivo imaging showed that both groups revealed fluores- nanoprobes by TP excitation. In line with this rationale, Fowley
cence from the glioma sites, but the intensity of An-PEG-Cdots et al. [81] designed a Cdots-protoporphyrin IX conjugate for use in
group was about 2-fold higher than that of PEG-Cdots group. TP-excited PDT (Fig. 8). Cdots not only converted the NIR light to
Moreover, the glioma/normal brain ratio (G/N ratio) showed that, visible light for excitation, but also increased water solubility of
compared to the ratio of 1.12 for PEG-Cdots group due to the EPR the PS by acting as a carrier. In vivo study showed that the growth of
effect, the ratio for An-PEG-Cdots group reached 1.73, semiquan- tumor size was only retarded in the presence of both light and
titatively indicating the improvement of glioma targeting capa- drug. Strictly relying on the presence of oxygen, conventional PDT
bility after the angiopep-2 modification. Prior to the potential has serious limitations in hypoxic conditions, which is typical
clinic translation of Cdots-based nanoformulations, many rele- for many solid tumors. To solve this problem, they covalently
vant issues are to be tested, one of which is the influence of attached a NO photodonor onto the surface of carbon dots to
injection routes. Huang et al. [77] thoroughly investigated the
effect of injection routes on the biodistribution, clearance, and
tumor uptake of carbon dots in mice. They predicted that carbon
dots owned great potential as a nanoplatform for preclinical
biomedical research such as optical imaging-guided theranostics.
With the involvement of an anticancer drug, Tang et al. [78]
developed a Cdots-based drug delivery system (DDS) featured by
FRET process and TP imaging. After PEGylation, the Cdots served
as scaffold to upload the drug molecule, i.e. doxorubicin (DOX)
with the protection from the PEG network and via p–p stacking.
FA was then covalently linked on the Cdots surface for targeting
cancer cells overexpressing FR. The DDS exhibited a pH-dependent
release behavior, mirrored by the concomitant change in FRET
signal. In contrast to free drug molecule, the DDS showed sus-
tained release over a much longer period. Single-photon cellular
imaging identified the effectiveness of this DDS in inducing apo-
ptosis of cancer cells. Moreover, owing to the large TP absorption
cross-section of Cdots, the TP imaging capability of the DDS was
further realized at deep tumor tissues of 65–300 mm under the
excitation of 810 nm (Fig. 7).
Photodynamic therapy (PDT) is an approved therapeutic treat-
ment for a number of cancers. In the presence of oxygen and light,
photosensitizer (PS) can transfer the absorbed photon energy to
surrounding oxygen molecules to form reactive oxygen species
(ROS) such as singlet oxygen (1O2) and free radicals, resulting in FIGURE 8
cell death and tissue destruction. However, many PS molecules are Schematic representation of the Cdots-protoporphyrin IX conjugate
not well soluble at aqueous conditions and characteristic of low showing indirect excitation of the protoporphyrin IX upon two-photon
biocompatibility. To tackle such problems, Huang et al. [79] excitation of the Cdots (top left). Photographs of Cdots-protoporphyrin IX
and protoporphyrin IX alone in phosphate buffer (top right). Plot of %
designed a PS-conjugated Cdots system featured by FRET for both
tumor growth after 4 days in tumor bearing mice that received the
enhanced fluorescence imaging and PDT therapy. Chlorin e6 (Ce6) following treatments: Cdots-protoporphyrin IX only (Drug only); two-photon
was selected as the PS, which emitted bright red fluorescence at irradiation at 800 nm (Light only); and Cdots-protoporphyrin IX and two-
668 nm upon excitation at 430 nm due to the efficient FRET process photon irradiation at 800 nm (Drug + Light). Reprinted with permission
with carbon dots. The feasibility of the system for fluorescence from Ref. [81]. Copyright 2013 Royal Society of Chemistry.

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Materials Today  Volume 19, Number 7  September 2016 RESEARCH

create Cdots-NO photoreleaser nanohybrids for TP phototherapy
of hypoxic tumors [82]. This NO-photostimulated treatment mo-
dality is expected to complement the ROS-generating PDT in clinic
treatment.
As a whole, carbon dots as the carrier for drugs or PSs confer
noteworthy advantages to theranostic bioimaging. First, the sta-
bility and solubility of drugs or PSs in aqueous/biological media
improve. Second, the cytotoxicity of drugs or PSs attenuates.
Third, the blood circulation of drugs or PSs prolongs. Fourth,
tumor-homing ability of drugs or PSs enhances due to the EPR
to Gd-DTPA, the Gd-Cdots exhibited much minimized cytotoxici-
ty. At 7 T, the longitudinal relaxivity (r1) of the Gd-Cdots was
5.88 mM1 s1 on a Gd basis, higher than that of Gd-DTPA
(3.10 mM1 s1) under the same conditions. Moreover, the Gd-
Cdots exhibited a high fluorescence quantum yield of 19.7%,
higher than that of Cdots (about 12%) obtained by calcining
DTPA. In vivo MRI with Gd-Cdots in normal nude mice showed
that the nanoprobe was excreted from the body via renal clear-
ance. For imaging tumor, a tumor targeting peptide, c(RGDyK),
was coupled onto the Gd-Cdots. T1-weighted MR images showed

effect and/or targeting effect. Last, carbon dots can excite drugs or
PSs indirectly by FRET mechanism.
Magnetic resonance imaging (MRI) is one of noninvasive imag-
ing techniques widely used in clinical diagnosis with high spatial
resolution and depth penetration. Gadolinium(III)-containing
contrast agents are frequently used in clinical MRI due to their
excellent contrast efficiency. Owing to the high toxicity of free Gd,
to encase Gd(III) into a nanoparticle capsule or carrier is one
approach that recently arouse great interests, but the sizes of these
nanoprobes should be strictly controlled, since large nanoparticles
tend to accumulate in organs of the RES, especially the liver and
spleen. Recently, Chen et al. [83] performed a detailed investiga-
tion of Gd(III)-doped carbon dots (Gd-Cdots) on the in vivo study
(Fig. 9). They prepared the Gd-Cdots by direct calcination of
gadopentetic acid (Gd-DTPA) at 300 8C for 2 h in air. Compared
RESEARCH: Review
that, after 4 h of injection, a signal enhancement of 42.6% was
observed in U87MG tumors of mice dosed with c(RGDyK)-Gd-
Cdots compared to those dosed with Gd-Cdots. Furthermore, the
high quantum yield of Gd-Cdots enabled the realization of immu-
nofluorescent staining areas to be observed overlapping with the
fluorescence emitting from Gd-Cdots. Other research groups pro-
posed different ways to synthesize Gd-Cdots nanoprobes with
varied r1 values and fluorescent quantum yields [84–86]. Such
differences may lie in the discrepancy in Gd doping as well as
the preparative methods.
Based on the ability of Cdots to absorb NIR light, Wu et al. [87]
first conducted photoacoustic (PA) imaging of sentinel lymph
nodes (SLN) in vivo by using Cdots passivated by macromolecules.
Observable signal enhancement of the SLN was exceptionally
rapid within 2 min after injection of the agent (Fig. 10). This

FIGURE 9
(a) T1-weighted transverse MR images. Gd-Cdots or RGD@Gd-Cdots (3.2 mg/kg) was intravenously injected into U87MG tumor bearing mice. Images were
acquired at 0, 10, 30, 45, 60 and 240 min. (b) T1-weighted coronal MR images. Significant signal enhancement was observed in tumors of animals injected
with RGD@Gd-Cdots. (c) Relative signal change at different time points based imaging results from (b). (d) Immunofluorescence histology study with tumor
samples. Red: integrin b3 (Cy5); blue: fluorescence from Cdots. Scale bars: 50 mm. Reprinted with permission from Ref. [83]. Copyright 2014 John Wiley &
Sons.

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RESEARCH: Review
FIGURE 10
Non-invasive real-time in vivo PA imaging of SLN in a nude mouse: For all
PA images, the laser was tuned to a wavelength of 650 nm. Control: PA
image acquired before injecting carbon dots. Red parts represent optical
absorption from blood vessels (BV); PA images acquired after the injection
of carbon dots. Blood vessel (BV), lymph vessel (LV), and sentinel lymph
node (SLN) are marked with arrows, and the SLN is visible between 2 and
90 min, but the contrast is much weaker after 210 min post-injection.
Reprinted with permission from Ref. [87]. Copyright 2013 Springer.
nanoprobe exhibited strong optical absorption in the NIR region,
small size, and rapid lymphatic transport, offering valuable po-
tential for faster resection of SLN and may lower complications
caused by using dyes or low-resolution imaging techniques. Most
recently, Ge et al. [88] prepared Cdots with red emission from
hydrothermal reaction of polythiophene phenylpropionic acid
(PPA). Displaying broad absorption in the visible to NIR region,
the Cdots could also be used as PA imaging agents. Furthermore,
the Cdots showed high photothermal conversion efficiency under
NIR light irradiation. Assisted by the bimodal imaging outcomes
(fluorescence + PA), they achieved photothermal ablation of xeno-
grafted tumor on nude mice with a 671 nm laser, demonstrating
the therapeutic effect of such Cdots. Cdots alone are hardly
considered as toxic to cells and tissues. Recently, Chang’s group
observed that the Cdots prepared from used green tea and ginger
juice exhibited surprisingly high inhibition activity toward cancer
cells through apoptosis while showed low toxicity to normal cells
[89,90]. All the examples imply the precursors to carbon dots are
significant for their theranostic utilities.
Gene therapy is regarded as a promising treatment for many
diseases. To accomplish it, a suitable carrier for safe and efficient
delivery of therapeutic payload into cells is required. Endowed with
excellent biocompatibility and unique fluorescent properties, car-
bon dots demonstrate great potential as a new family of carriers for
gene delivery. Liu et al. [91] first employed PEI (bPEI25k) passivated
Cdots as a transfection agent. These positively charged Cdots
exhibited good water solubility and bright fluorescence, manifest-
ing in vitro DNA transfection efficiency comparable to control PEI,
but with lower cytotoxicity. Inspired by this study, Kim et al. [92]
proposed a ternary complex comprising PEI-functionalized carbon
dots (PEI-Cdots), PEI-functionalized gold colloids (PEI-Au), and
plasmid DNA (pDNA) for in vitro DNA transfection and dynamic
monitoring of plasmid cellular trafficking. Initially the complex had
weak fluorescence emission due to the connection of PEI-Cdots with
PEI-Au by the role of the anionic pDNA. At the intracellular level
during transfection especially post-endosomal step when high salt
concentration exists, PEI-Cdots would dissociate from PEI-Au by the
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Materials Today  Volume 19, Number 7  September 2016
release of pDNA and fluorescence recovery, enabling gene delivery
and monitoring of its cellular trafficking concomitantly. Most
recently, Wang et al. [93] and Pierrat et al. [94] individually demon-
strated the utility of Cdots to mediate in vivo transfection.
Summary and outlook
After a decade of extensive researches, the initially emergent nano-
lights of carbon dots have developed to one of the most valuable and
intriguing groups of nanomaterials nowadays. The number of sci-
entific papers on carbon dots is increasingly expanded each year
with the orientation shifting from synthetic methods and cellular
optical imaging in early days to fabrication of multifunctional
carbon dots-based nanohybrids presently with promising applica-
tions in health, environment, and energy. There are several reasons
responsible for the public interests on carbon dots. First, the syn-
thesis is generally simple and economic by selecting precursors from
a wide spectrum of cheap carbon sources. Second, further surface
functionalization is adaptable and convenient. Third, they are
endowed with unique physical properties, excellent aqueous stabil-
ity, and outstanding biocompatibility. Fourth, their complex with
other species often results in improved performance.
The most fundamental thing is the synthesis of carbon dots. Even
though a large number of synthetic approaches have been devel-
oped, those with the advantages of eco-friendliness and low cost are
preferably needed. This means more when pertaining to large-scale
synthesis for technological purpose. To date, most of works claiming
large-scale synthesis report the production of carbon dots at gram
scale, which needs to be expanded in future studies. Along with the
enlarged preparation, reproducibility in physical properties of car-
bon dots should be taken into serious consideration.
Photoluminescence is still the mostly interested property of
carbon dots, and the mechanism underlying it is still in debate.
Such discrepancy is partly caused by the difference in preparation
methods or use of different carbon precursors. With respect to
carbon dots derived from raw carbon sources, the emission is now
increasingly believed to be due to radiative recombination of
surface-confined electrons and holes. And for carbon dots pro-
duced from biomass and molecules, highly emissive fluorophores
left on the carbon dots surface are assumed to be responsible for
the observed bright fluorescence.
Possibly by structure, nearly all of carbon dots show maximum
fluorescence emission in blue or green spectra region, irrespective
of the carbon sources or preparation methods. Until now, many
carbon dots possess fluorescence quantum yields comparable to
those of metal-based semiconductor quantum dots, enabling them
potential alternatives to quantum dots in areas where better
biocompatibility is desired. For sensing applications, a lot of
sensors based on bare carbon dots have been developed, and
the next step is to add more functional groups to carbon dots
for better target selectivity. In fact, carbon dots can substitute
metal-based quantum dots in many sensing platforms developed
at present since they can be adapted to share similar surface groups
quite simply. For optical imaging of cells, carbon dots enter into
cells mainly through passive endocytosis and label the perinuclear
cytoplasm predominantly. For a better accumulation of carbon
dots in the cell, it is advisable to couple membrane translocation
agents; and for specific labeling of organelles, ligands with target-
ing capability should be employed. Several seminal works have
Materials Today  Volume 19, Number 7  September 2016 RESEARCH

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[45] J.X. Shi, et al. Biosens. Bioelectron 45 (2013) 58.
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21407140, 21431006, 91022032), and the Foundation for [51] J. Zhang, L. Dong, S.H. Yu, Sci. Bull. 60 (2015) 785.
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