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Nano Res
1
One step synthesis of fluorescent smart
thermo-responsive copper clusters: a potential
nanothermometer in living cells
Chan Wang a,c, Lin Ling a,b, Yagang Yao b(), Qijun Song a()
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0707-0
http://www.thenanoresearch.com on January 6, 2015
© Tsinghua University Press 2015
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Nano Research
DOI 10.1007/s12274-015-0707-0
One step synthesis of fluorescent smart
thermo-responsive copper clusters: a potential
nanothermometer in living cells
Chan Wang a,c, Lin Ling a,b, Yagang Yao b, Qijun Song a
a Jiangnan University, China.
b Suzhou Institute of Nano-tech and Nano-bionics,
Chinese Academy of Sciences, China.
c Nanjing Tech University, China.
Highly luminescent and stable CuNCs are synthesized by a green and
convenient route, displaying a smart and reversible response upon
temperature cycles. The attractive thermal feature allows the CuNCs to
serve as thermal-responsive functional material.
One step synthesis of fluorescent smart
thermo-responsive copper clusters: a potential
nanothermometer in living cells
Chan Wang a,c, Lin Ling a,b, Yagang Yao b(), Qijun Song a()
Received: day month year
Revised: day month year
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
fluorescence,
copper nanoclusters,
cellular imaging,
nanothermometer
ABSTRACT
Temperature measurement in biology and medical diagnostics, and sensitive
temperature probe in living cells, is of great importance, however, is still a
challenge. Metal nanoclusters (NCs) having attractive luminescent properties
may be a promising candidate to deal with such challenge. Here, a novel one
step synthetic method is presented for the preparation of highly fluorescence
copper nanoclusters (CuNCs) in ambient condition using glutathione (GSH) as
both the reducing agent and the protective layer preventing the as-formed NCs
from aggregation. The resultant CuNCs contain 13 atoms with an average
diameter of 2.3 nm and exhibit red fluorescence (λEm = 610 nm) with high
quantum yields (QYs, up to 5.0%). Interestingly, the fluorescence signal of
CuNCs is reversibly responsive to the environmental temperature from 15 to
80 °C. Furthermore, the CuNCs exhibit good biocompatible, which can enter
into MC3T3-E1 cells, and enable the measurements over the physiological
temperature range 15–45 ºC by using confocal fluorescence imaging method. In
view of the facile synthesis method and attractive fluorescence properties, the
as-prepared CuNCs could be used as a photoluminescence thermometer and
biosensor.
1 Introduction
Temperature is one of the most frequently measured
parameters that governs biological reaction within
living cells [13]. The accurate measurement of
temperature and its gradient inside a living cell can
promote the advancements in cell biology and
biomedicine [4]. However, the conventional
temperature sensors, i.e., thermocouples, thermistors
and infrared thermometers, could not meet the
requirements of measurement in living cells [5, 6].
For instance, thermocouples are unable to function
within cells, and can only be used in the contact with
the testing surrounding, making the operation
Nano Research
DOI (automatically inserted by the publisher)
Address correspondence to Q. Song, [email protected]; Y. Yao, [email protected].
Research Article
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2 Nano Res.
complicated and low spatial resolution. Infrared
thermometers based on blackbody radiation can only
measure the surface temperature of the materials,
and the infrared light is easy to be absorbed when
the light penetrated through some medium like
steam and glass [7]. Therefore, the increasing
expectations in monitoring cell temperature greatly
call for novel nanothermometers [8]. In this respect,
fluorescence-based temperature sensors have shown
great potential since they operate as “non-contact”
tools and offer the dual function of cellular imaging
and temperature sensing at the molecular level [2, 6,
9]. Many promising fluorescent materials, such as
quantum dots [10, 11], rare earth-doped [12, 13] and
polymeric hydrogel nanoparticles [14], are being
widely used at present.
Fluorescent metal nanoclusters (NCs) composing
of several to tens of atoms have gained extensive
attentions. Relative to their larger counterparts
(nanoparticles), metal NCs possess size comparable
to Fermi wavelength of electrons, and exhibit unique
molecular-like properties, such as well-defined
molecular, HOMO-LUMO transition, molecular
magnetism, and strong luminescence. [1518]. In the
past decades, many researches focused on the
development of fluorescent AuNCs and AgNCs as
the ideal fluorescent labels for biological applications,
owing to their attractive features, such as chemical
stability, excellent photostability, ultrasmall size and
good biocompatibility [1922]. Compared with Au
and Ag, non-precious Cu is earth-abundant and
significantly cheaper, and Cu nanoclusters (CuNCs)
possess unique photoluminescence (PL) properties
[23, 24]. Despite these advantages, CuNCs suffered
from the difficulties in controlling their ultrafine size
and the susceptibility to the oxidation upon exposure
to air [25, 26]. Therefore, studies on the preparation
of CuNCs are still in a preliminary stage [27].
Currently, the preparations of fluorescent CuNCs
were mainly depended on the bottom-up method,
which were based on the chemical reduction of metal
ions to form metal atoms, and then CuNCs were
produced via the accumulation of metal atoms.
However, these methods were complicated and
time-consuming, causing low quantum yields (QYs)
due to the aggregation of CuNCs. To circumvent this
problem, the protective layer such as polymers, voids
in zeolites or other microporous solids has been
employed to prepare small metal NCs [28, 29], but
the processes were still complicated, which may limit
the usage. Here, we report a new one-step method to
prepare fluorescent CuNCs by using glutathione
(GSH) as both the reducing agent and the protective
layer. Intriguingly, the resultant CuNCs exhibit
strong luminescence, good biocompatibility and
smart response to the external temperature. The
attractive features allow the CuNCs to serve as
thermal-responsive functional materials. Hence, we
explore the utility of the GSHCuNCs for cellular
imaging and intracellular temperature
measurements.
2 Experimental
2.1 Materials
Reduced glutathione (GSH, molecular weight of 307),
3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium
bromide (MTT), Dimethyl sulfoxide (DMSO) and
Rhodamine 6G (cat. no. 252433) were purchased
from Sigma-Aldrich. Copper nitrate (Cu(NO3)2) and
alcohol were analytical grade. MC3T3-E1 cells were
available in the Cell Bank of Type Culture Collection
of Chinese academy of sciences. Cell culture products
and reagent, unless mentioned otherwise, were
purchased from GIBCO. All reagents were used as
received without further purification. Deionized
water was used in all experiments.
2.2 CuNCs synthesis and optimization
The important reaction parameters including the
mole ratio of GSH to Cu(NO3)2, reaction temperature,
and reaction time were investigated to obtain the
GSH-stabilized CuNCs. Here the reaction time
means the soaking time after heating to reaction
temperature.
In a typical procedure, GSH (31 mg) and Cu(NO3)2
(6 mg) were added to 5 mL deionized water. The
solution was stirred at room temperature for a certain
time, and a white hydrogel was formed due to the
coordination between Cu ions and functional groups
of GSH (i.e. –NH2, –COOH, –SH). The hydrogel was
heated at 80 ºC under stirring, and the reaction time
was 10 min. After that, the NaOH solution (1 M) was
dropwise added until a light yellow and transparent
solution was obtained, and the corresponding pH
value was 45. After cooling to room temperature,
the products were collected by precipitating with
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3 Nano Res.
alcohol and centrifugation at 6000 rpm. The above
purification process was repeated for three times,
followed by collecting the precipitate on the bottom.
The resultant GSHCuNCs were freeze-dried under
the vacuum, and stored in the refrigerator for
long-term preservation.
2.3 Quantum yield Calculations
The QY of the as prepared GSHCuNCs was
obtained using a 502 nm Xe laser and calibrated with
Rhodamine 6G dissolved in ethanol (QY = 95%).
According the emission peak area and absorbance of
GSHCuNCs and Rhodamin 6G, the QY of the
GSHCuNCs could be calculated from Equation 1
below: [30]
Where Φstd is the quantum yield of the standard
compound, Fsample and Fstd are the integrated areas of
fluorescence of the sample and standard in the
emission region at 450–750 nm. Astd and Asample are the
absorbance of the standard and sample at the
excitation wavelength (410 nm); is the refractive
index of solvent, for water the refractive index is 1.33,
and ethanol is 1.36. All samples were diluted to
ensure the optical densities less than 0.07 measured
by Varian Cary 50 UV-Vis spectrophotometer to
reduce the error.
2.4 Cell culture and MTT assay
MC3T3-E1 cells were cultured in Dulbecco's
modified eagle medium (DMEM, HyClone) onto a
96-well plate (Corning, Costar, NY) for 12 h,
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin antibiotic (100 U of penicillin
and 100 g/mL streptomycin sulfate) at 37 ºC in a
humidified incubator containing 5% CO2. For the
MTT cell viability assay, cells in a 24-well plate were
incubated with various concentrations of CuNCs (10,
20, 40 and 80 g/mL) for 24 h. After exposure, the
supernatant was removed and cells were washed
immediately with PBS. Then, an aliquot of 150 μL
DMEM and 15 μL MTT stock solutions (5 mg/mL in
PBS, pH 7.4) were subsequently added to each well
and incubated for 4 h at 37 C, followed by removing
the culture medium with MTT, and then 150 μL
DMSO was added.
The resulting mixture was shaken for ca. 5 min at
room temperature. The optical density (OD) of the
mixture was measured at 490 nm using a standard
micro plate reader (Scientific Multiskan MK3, thermo,
USA), and the cell viability was estimated according
to the following Equation 2: [30]
Where ODControl was obtained in the absence of
CuNCs, and ODTreated was obtained in the presence of
CuNCs.
2.5 Cell imaging
Primary GSHCuNCs solution (20 g/mL) was
dispersed into cells in secrum-free DMEM and
incubated for 2 h at 37 ºC in the presence of 5% CO2.
The cells in the culture medium were then washed
three times with warm PBS to remove the excess
nanoclusters in advance, and added the fresh culture
medium. After that, the sample temperature was
adjusted by a heater, and three replicate samples
were prepared for each temperature point. To avoid
the cells dead, the cells in the culture medium were
directly detected under the confocal fluorescence
microscope, and this process must be operated
quickly. Prior to inspection, the temperature was
taken and recorded by the thermometer.
2.6 Characterization
UV-Vis absorption spectra were recorded with a
Lambda 800 spectrophotometer (PerkinElmer, USA).
Photoluminescence (PL) experiments were carried
out with a Shimadzu RF-5301 PC spectrofluorimeter
(Shimadzu, Japan), with excitation at 400 nm. X-ray
photoelectron spectroscopy (XPS) analysis was
performed on a VG ESCALAB MKII spectrometer
(ThermoFisher Scientific, USA) with Mg K
excitation (1253.6 eV), and the binding energy was
calibrated with the C 1s band at 284.6 eV. The
following sequence of spectra was recorded: N 1s, O
1s, C 1s and S 2p, where the C 1s was recorded for
two times to check the instrument stability and the
possible sample degradation during analyses.
Electrospray ionization time of flight mass
spectrometric (ESITOF MS) studies were carried out
on a Xevo G2-S Tof MS spectrometer (Waters, USA),
and the data were collected in the negative ion mode
(%) = ( ) 100% 2Treated
Control
ODCell Viability
OD ( )
2
2= 1
sample samplestdsample std
sample std std
FA
A F
()
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4 Nano Res.
with a full scan over the range of 501400 m/z.
Fourier transform infrared spectroscopy (FT-IR)
spectra were recorded in 4000500 cm1 with a
Nicolet Avatar 360 FT-IR spectrophotometer
(ThermoFisher Scientific, USA). Transmission
electron microscopy (TEM) analyses were
characterized on TECNAI F20 (FEI, USA) to study
the morphology and mean diameter of the resultant
GSH–CuNCs, operating at an accelerating voltage of
200 kV. The confocal microscopy images were
observed under a confocal fluorescence microscope
(FV1000, Olympus) at 37 ºC (INUB-ONICS, Tokai
Hit). All measurements were performed at room
temperature (25 ºC).
3 Results and discussion
3.1 Synthesis of CuNCs
Since the existence of a thiol group in the molecule
structure of GSH, it has the intrinsic metal-chelating
properties to ensure the formation of high-affinity
metalligand clusters [3133]. Apart from this, thiol
groups are able to etch larger NCs or NPs to reduce
their sizes and improve their sizes monodispersity
[34]. Moreover, the groups of carboxyl and amino in
GSH molecule tend to provide a protective layer,
ensuring the stability of fluorescence properties of
CuNCs. More importantly, recently GSH has been
demonstrated as the reducing agent for the synthesis
of nanoparticles due to the existence of amino groups
[17, 34, 35]. Therefore, GSH was chosen in this study
to serve as both the protective layer and the reducing
agent. By simply mixing GSH with copper source in
aqueous solution, a one-step process was developed
to prepare CuNCs. It is worth mentioning that there
was no need to control the end-point by pH value of
the solution, and the NaOH solution was slowly
added until a light yellow and transparent solution
was obtained. Three important reaction parameters
including the molar ratio of GSH/Cu, the reaction
temperature and time were optimized to improve the
PL intensity of CuNCs, which will be discussed later.
As literature reported [36], formation of metal NCs
involved two steps, and the first step was the
reduction of Cu(II) to Cu(I) or Cu(0) by GSH,
followed immediately by the coordination of Cu(I) or
Cu(0) to the thiol group in GSH to produce insoluble
colloid of Cuthiolate complexes. The second step
initiated by addition the NaOH was the dissolution
of the colloid of Cuthiolate complexes, and the
conversion to stable CuNCs [37]. To determine the
optimal GSH/Cu molar ratio, various GSH contents
(GSH/Cu = 1:1, 2:1, 4:1, 5:1, mol/mol) were
investigated and the corresponding PL spectra were
recorded as shown in Fig. 1(a). When the GSH
content was low, the reaction between GSH and Cu(II)
was the rate-controlling step, and the solution was in
yellow. With the increase of the GSH/Cu ratio, the
fluorescence intensity increases up to a maximum at
GSH/Cu = 4, and the thermodynamically stable
GSH–CuNCs were obtained. The solution color
changed to light yellow. Afterwards, the intensity
decreases. This is probably because that once the
GSH content is excess, more of free GSH molecules
are in the solution, which will result in cloudy
solution and fluorescence quenching of CuNCs,
owing to the possible formation of metal complexes
through the reaction of free GSH and CuNCs [38,39].
The visualized photographs of the resultant solution
about different GSH/Cu ratio are shown in Fig. S1.
Therefore, the optimal GSH/Cu molar ratio is 4.
The effects of the reaction temperature and time on
PL intensity of CuNCs were also investigated, and
the corresponding results were presented in Fig. 1(b)
and (c), respectively. Apparently, at lower reaction
temperature the yield of CuNCs will be relatively
lower due to lower reactivity of GSH, and thus a
decreased fluorescence intensity was observed
shown in Fig. 1(b). The highest fluorescence intensity
was achieved at the reaction temperature of 60 °C.
With further increasing the temperature, the reaction
became so fast that the resultant CuNCs tended to
agglomerate, consequently a decrease in fluorescence
intensity was observed. As for the effect of the
reaction time, the PL intensity increased with the
increase of the reaction time in the range of 0 to 10
min as demonstrated in Fig. 1(c). Prolonged reaction
time to 30 min caused the rapid decrease of the
fluorescence intensity, indicating the formation of
large nonfluorescent Cu nanoparticles [40]. Therefore,
we set the reaction time of 10 min.
3.2 Characterization of CuNCs
According to the above experiments, we synthesized
the GSH-protected CuNCs at 60 °C for 10 min with
the molar ratio of GSH to Cu(NO3)2 at 4. The UV-Vis
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5 Nano Res.
and PL spectra of the resultant CuNCs are shown in
Fig. 2. The as-prepared CuNCs exhibit good
dispersion in aqueous solution with no obvious
precipitation, as the protective layer of GSH can
prevent the agglomeration of nanoclusters. The
CuNCs show a color of light yellow under ambient
light, and a bright red fluorescence under UV
irradiation (see inset of Fig. 2(a)). In addition, the
characteristic absorption peak at 507 nm, arising
from the surface plasmonic resonance of Cu
nanoparticles, were not observed in UV-Vis spectra,
instead strong absorbance peaks were found in the
wavelengths below 300 nm, indicating the formation
of CuNCs [41, 42]. As shown in Fig. 2(b), the CuNCs
exhibit the red emission with a peak at 610 nm and
corresponding full width at half maximum (FWHM)
around 90 nm under the excitation peak of 410 nm.
The quantum yield of GSH–CuNCs in aqueous
solution at room temperature is found to be 5.0%
using rodamine 6G (QYs, 0.95 in ethanol) as the
standard, which is much higher than that of CuNCs
prepared in the previous work [31].
To directly view the CuNCs, the TEM analysis was
performed, and the results are displayed in Fig. 3.
The CuNCs are uniformly dispersed, and possess an
average diameter of about 2.3 nm in the range of
1.53.0 nm, without large metal nanoparticles or
aggregation (seen in Fig. 3(a)). The XPS analysis was
carried out to determine the oxidation state of copper
in the GSH–CuNCs. As shown in Figure 3b, two
peaks appear at 932.1 and 953 eV, which can be
ascribed to the binding energies of the 2p3/2 and
2p1/2 electrons of Cu(0), respectively [43]. The
absence of Cu 2p3/2 satellite peak around 942.0 eV
confirms that there is no existence of Cu(II) electrons
[25, 44]. Noting that the binding energy of Cu(0) is
only 0.1 eV away from that of Cu(I) [45], so it is not
possible to exclude the formation of Cu(I), and the
valence state of the obtained CuNCs most likely lies
between 0 and +1. As reported in literatures [37, 46],
the metal(0)@metal(I)thiolate coreshell NCs
exhibited strong luminescence. Moveover, the Cu
atoms in such tiny clusters were expected to be
positively charged, and the existence of Cu(I) could
have contributed to the enhancement of both stability
and PL intensity of CuNCs [47].
Despite the great progress in synthesis and
characterization, the mechanism studies of NCs
formation still significantly lag behind. The
fluorescence metal NCs with precise molecular
formula helps for understanding unresolved
luminescence fundamentals [48]. To determining the
cluster formula of metal NCs, the electrospray
ionization time of flight mass spectrometry (ESI-TOF
MS) is a well-accepted technique [21, 49], and the
representative results about CuNCs are shown in Fig.
4. The peak at m/z ≈ 613.1 may be assigned to the
oxidized GSH. With the help of the isotopic mass
distribution of Cu, the highest peaks at m/z ≈ 802.9
can be assigned to the Cu cluster with a composition
of Cu3L2 (L = C10H16O6N3S), whereas those in the
lower mass range may be ascribed to the fragments
of Cu2L2 (m/z ≈ 739.0), Cu1L2 (676.0) and Cu1L1 (370.0).
Among these, Cu1 and Cu2 clusters are the dominant
Cu-containing components in the colloid solution.
The extensive theoretical studies have predicted the
existence of stable Cun clusters with n = 19 [50, 51].
The CuNCs shown by TEM images are much larger
than those obtained from MS analysis, and this is
because the larger CuNCs observed in the TEM
images are difficult to be ionized, so that they could
be not detected in the MS, where only the small
molecule fragments are detected [52].
Further chemical and surface properties of CuNCs
were exploited by fourier transform infrared
spectroscopy (FT-IR) measurements (Fig. S2). The
GSH exhibits a number of characteristic IR bands, i.e.,
COO (1390 and 1500 cm1), the NH stretch (3410
cm1) and the NH bending (1610 cm1) of NH2. The
peak observed at 2526 cm1 can be assigned to the
SH stretching vibrational mode, which disappeared
completely in CuNCs, suggesting the cleavage of the
SH bond and the binding of the GSH molecules
onto the surface of CuNCs through CuS bonding.
The XPS spectra of other elements (i.e., S 2p, C 1s, N
1s and O 1s) are shown in the Fig. S3. The C1s peak
could be disintegrated into four different
components at 288.5 eV (COOH), 287.8 eV
(CONH2), 285.4 eV (CH) and 284.6 eV (CH2CH3)
[53]. Three peaks at 161.9, 163.1 and 168.4 eV are the
characteristic signals of S 2p, which could be
assigned to the CuS bond, elemental and oxidation
state of sulfur, respectively. The N 1s peaks at 399.5
and 401.1 eV indicate the presence of –NH and –NH3+
in GSH molecules, respectively [54]. The peak at
531.1 eV is in accordance with the binding energy of
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6 Nano Res.
O 1s.
3.3. Thermoresponsive properties of CuNCs
To explore the potential applications of the obtained
CuNCs for intracellular nanothermometry, we
investigated their thermoresponsive to the external
environment. The PL spectra were measured with a
series of CuNCs samples at the temperature ranged
from 15 to 80 °C. The measurements were conducted
in both forward and backward temperature mode to
ensure the reproducibility, and the results are
demonstrated in Fig. 5. When the temperature rises
from 15 to 80 °C at a step of 5 °C, the emission
intensity of CuNCs decreases almost 85%, while the
emission spectra of CuNCs do not shift within the
investigated temperature window (Fig. 5(a)).
Afterwards, when the temperature decreases back
from 80 to 15 °C, the emission intensity was fully
recovered (Fig. 5(b)). This is a typical characteristic of
the temperature-sensitive fluorescence materials,
which follows Boltzmann distribution, that is, as
temperature increases, the molecules collision
frequency and the nonradiative transition rate
increases, while radiative transition rate is constant,
decreasing the intensity of emission from the excited
state (i.e. the fluorescence intensity). Conversely, low
temperature is beneficial to increase the fluorescence
intensity of CuNCs [55].
Fig. 5(c) summarizes the temperature effect on the
PL intensity of CuNCs, and the data were obtained
from one temperature cycle shown in Fig. 5(a) and
(b). The repeatability of the emission intensity
measured at different temperatures indicates that the
CuNCs have excellent stability to the temperature
variations. To further investigate the reproducibility
of CuNCs, the luminescence switching operations
were repeated for five consecutive cycles by multiple
heating and cooling cycles between 15 and 70 °C. The
results are presented in Fig. 5(d), and the
corresponding spectra collected are shown in Fig. S4.
At 15 °C, the emission intensity is almost constant
and higher than that obtained at 70 °C, which follows
the same tendency as conventional fluorophores [56].
Obviously, our CuNCs exhibit
temperature-dependent PL intensity without fatigue,
indicating a good reversibility of the two-way
switching processes. The new-discovered smart
fluorescence features of CuNCs discussed above,
together with their good dispersibility in water, red
fluorescence and high QYs, enable the CuNCs
applicable as PL thermometers and biosensors in cell
monitoring.
3.4. CuNCs for cell imaging and intracellular
nanothermometer
Although excessive amount of copper is harmful to
living organism, low amount of copper could play a
pivotal role in many fundamental physiological
processes [57]. To evaluate the cytotoxicity of CuNCs,
a thiazoyl blue tetrazolium bromide (MTT) assay was
conducted to the cells loaded with CuNCs prior to
the experiments. From Fig. 6, the viability of
MC3T3-E1 cells remain above 80% after CuNCs
exposure even at the concentration of 80 μg/mL for
48 h, and these conditions are much vigorous than
that used for cell incubation and imaging. The results
confirm that our CuNCs show an excellent
biocompatibility and have no adverse effect to
MC3T3-E1 cells. Meanwhile, the intracellular
distribution of the CuNCs was evaluated by confocal
laser fluorescence microscopy, and the images are
shown in Fig. S5. From the bright-field image, it is
apparent that MC3T3-E1 cells maintain their
morphology after incubated with CuNCs at the
specific dose and time. Besides, the fluorescent
signals are not only detected in the cytoplasm but
also in the cellular nucleus, as demonstrated in Fig.
S5(b), indicating the feasibility of CuNCs for cellular
imaging. To further exemplify, the Z-scanning
confocal fluorescence microscopy images was
performed to demonstrate the intracellular
internalization of CuNCs, which were taken from one
side to the other side of the cell, and the results were
exhibited in Fig. S6. The GSHCuNCs had entered
into living cells, not just adsorbed on the surface of
the cells.
The stability of CuNCs in PBS medium was
evaluated for blank control, and the data was
displayed in Fig. S7. The fluorescent CuNCs
exhibited thermoresponsive to the CuNCs/PBS
solution, of which the trend was same as the CuNCs
in aqueous solution. We explore the capability of
resultant fluorescence CuNCs thermometers to
monitor intracellular temperature differences in
MC3T3-E1 human cancer cells using laser-scanning
confocal microscopy, and three samples under
different environmental temperature were tested as
shown in Fig. 7. As expected, the images provide
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7 Nano Res.
clear evidence that the fluorescence intensity
decreases markedly with the increase of temperature,
and the corresponding changes are easy to be
observed with the naked eyes. The above results
illustrate the great potential of our CuNCs for
sensing temperature at the subcellular level.
4 Conclusions
We present a convenient method with one step to
prepare highly fluorescent and stable CuNCs using
GSH as both the reducing agent and the protective
layer. The resultant CuNCs show remarkable
features including water-soluble, bright red
fluorescence and high QYs. Significantly, the
fluorescence signal of CuNCs is reversibly responsive
to external environmental temperature with good
reproducibility. According to the MTT assay,
GSHCuNCs can enter into cellular nucleus,
exhibiting good biocompatibility and providing the
possibility for cellular imaging. Moreover, our
CuNCs have the capacity for monitoring intracellular
temperature differences (1545 °C) in MC3T3-E1
human cancer cells using laser-scanning confocal
microscopy. Given the facile synthesis method and
attractive fluorescence properties, the prepared
GSHCuNCs are quite promising in applications of
imaging and sensing in living cells.
Acknowledgements
This work was supported by Natural National
Science Foundation of China (No. 51372265 and No.
21175060), the Natural Science Foundation of Jiangsu
Province, China (No. BK20140392), the Open
Foundation of State Key Laboratory of
Materials-Oriented Chemical Engineering of Nanjing
University of Technology (2014, KL14-12), the
Postdoctoral Research Foundation of Jiangsu
Province, China (No. 1401058B), and the Science and
Technology Project of Suzhou, China (No.
ZXG201428 and No. ZXG201401).
Electronic Supplementary Material: Supplementary
material (details of Fig. S1 to S6) is available in the
online version of this article at
http://dx.doi.org/s10.1007/.
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10 Nano Res.
Figure captions
Figure 1 Effect of (a) GSH content, (b) heating
temperature and (c) reaction time on the PL
intensity of the resultant CuNCs.
Figure 2 (a) UV-Vis absorption of GSHCuNCs
in aqueous solution; inset: photographs of the
GSHCuNCs under the irradiation of visible (left)
and UV (right) light; (b) Excitation (Ex) and
emission (Em) spectra of the resultant
GSHCuNCs.
Figure 3 (a) TEM micrograph (inset: size
distribution) of the synthesized fluorescence
GSHCuNCs; and (b) XPS spectra of Cu 2p in
GSHCuNCs.
Figure 4 Representative ESI-TOF MS spectrum
of a copper cluster sample detected in the
negative-ion mode.
Figure 5 Temperature dependence of the emission
intensity from GSHCuNCs in aqueous solution. (a)
Fluorescence emission spectra measured under
excitation.
Figure 6 Viability of MC3T3-E1 cells in cell
medium as the function of concentration, as
determined by an MTT assay. The error bars
represent variation among three independent
measurements.
Figure 7 Typical confocal fluorescent images of a
MC3T3-E1 cell with incorporated GSHCuNCs at
three different temperatures.
Figure 1 Effect of (a) GSH content, (b) heating temperature and (c) reaction time on the PL intensity of the resultant CuNCs.
Figure 2 (a) UV-Vis absorption of GSHCuNCs in aqueous solution; inset: photographs of the GSHCuNCs under the
irradiation of visible (left) and UV (right) light; (b) Excitation (Ex) and emission (Em) spectra of the resultant GSHCuNCs.
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11 Nano Res.
Figure 3 (a) TEM micrograph (inset: size distribution) of the synthesized fluorescence GSHCuNCs; and (b) XPS
spectra of Cu 2p in GSHCuNCs.
Figure 4 Representative ESI-TOF MS spectrum of a copper cluster sample detected in the negative-ion mode.
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12 Nano Res.
Figure 5 Temperature dependence of the emission intensity from GSHCuNCs in aqueous solution. (a) Fluorescence
emission spectra measured under excitation of 400 nm with the increase of temperature from 15 to 80 °C at a step of 5 °C
(from top to bottom); (b) Fluorescence emission spectra measured under excitation of 400 nm with the decrease of
temperature from 80 to 15 °C (from bottom to top); (c) Plots of PL intensity with temperature (integrated from 600 to 610 nm
during one temperature cycle); (d) Change of the PL intensity with five thermal cycles when the temperature increases
directly from 15 to 70°C and then back to 15 °C, of which the corresponding spectra collected shown in Fig. S3.
Figure 6 Viability of MC3T3-E1 cells in cell medium as the function of concentration, as determined by an MTT assay. The error
bars represent variation among three independent measurements.
Figure 7 Typical confocal fluorescent images of a MC3T3-E1 cell with incorporated GSHCuNCs at three different temperatures.
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Electronic Supplementary Material
One step synthesis of fluorescent smart
thermo-responsive copper clusters: a potential
nanothermometer in living cells
Chan Wang a,c, Lin Ling a,b, Yagang Yao b(), Qijun Song a()
Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)
Figure S1 Visualized photographs of the resultant solution about different GSH/Cu ratio. When the GSH
content was low, the reaction between GSH and Cu(II) was the rate-controlling step, and the solution was in
yellow. With increasing the GSH/Cu ratio to 4, the thermodynamically stable GSH–CuNCs were obtained, and
the solution changed to light yellow. Nevertheless, once the GSH content is excess, more of free GSH molecules
are in the solution, which will result in cloudy solution and fluorescence quenching of CuNCs, owing to the
possible formation of metal complexes through the reaction of free GSH and CuNCs.
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Figure S2 FT-IR spectra of the GSH and GSHCuNCs.
Figure S3 Typical XPS spectra of (a) C 1s, (b) S 2p, (c) N 1s and (d) O 1s involved in the resultant GSHCuNCs.
Address correspondence to Q. Song, [email protected]; Y. Yao, [email protected].
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Figure S4 PL spectra of GSHCuNCs in aqueous solution during five heating and cooling cycles between 15 °C
and 70 °C (integrated from 600 to 610 nm).
Figure S5 (a) Fluorescence, (b) bright field, and (c) overlay of fluorescent and bright field images of MC3T3-E1
cells incubated with GSHCuNCs, of which luminescence was in NIR region with a λEm = 610 nm for 24 h.
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Figure S6 The Z-scanning confocal fluorescence microscopy images of a MC3T3-E1 cell with incorporated
GSHCuNCs. To view the intracellular internalization of CuNCs, the images from S1 to S12 were taken from
one side to the other side of the cell by the confocal fluorescence microscopy. Obviously, the GSHCuNCs have
entered into living cells, not just adsorbed on the surface of the cells.
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Figure S7 PL spectra with a λEx = 410 nm of the CuNCs in PBS medium. When the temperature rises from 20 to
45 °C at a step of 5 °C, the emission intensity of CuNCs decreases, while the emission spectra of CuNCs do not
shift within the investigated temperature window.