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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




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.





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

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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

（）


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


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


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


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/.

References [1] Okabe, K.; Inada, N.; Gota, C.; Harada, Y.; Funatsu, T.;

Uchiyama, S. Intracellular temperature mapping with a

fluorescent polymeric thermometer and fluorescence

lifetime imaging microscopy. Nat. Commun. 2012, 3,

705–713.

[2] Jaque, D.; Vetrone, F. Luminescence nanothermometry.

Nanoscale 2012, 4, 4301–4326.

[3] McLaurin, E. J.; Bradshaw, L. R.; Gamelin, D. R.

Dual-emitting nanoscale temperature sensors. Chem. Mater.

2013, 25, 1283–1292.

[4] McCabe, K. M.; Hernandez, M. Molecular thermometry.

Pediatr. Res. 2010, 67, 469–475.

[5] Wolfbeis, O. S. Sensor paints. Adv. Mater. 2008, 20,

3759–3763.

[6] Lee, J.; Kotov, N. A. Thermometer design at the nanoscale.

Nano Today 2007, 2, 48–53.

[7] Ring, E. F. The historical development of temperature

measurement in medicine. J. Infrared Phys. Technol. 2007,

49, 297–301.

[8] Löw, P.; Kim, B.; Takama, N.; Bergaud, C.

High-spatial-resolution surface-temperature mapping using

fluorescent thermometry. Small 2008, 4, 908–914.

[9] Donner, J. S.; Thompson, S. A.; Kreuzer, M. P.; Baffou, G.;

Quidant, R. Mapping intracellular temperature using green

fluorescent protein. Nano Lett. 2012, 12, 2107–2111.

[10] Maestro, L. M.; Jacinto, C.; Silva, U. R.; Vetrone, F.;

Capobianco, J. A.; Jaque, D.; Solé, J. G. CdTe quantum dots

as nanothermometers: towards highly sensitive thermal

imaging. Small 2011, 7, 1774–1778.

[11] Albers, A. E.; Chan, E. M.; McBride, P. M.; Ajo-Franklin,

C. M.; Cohen, B. E.; Helms, B. A. Dual-emitting quantum

dot/quantum rod-based nanothermometers with enhanced

response and sensitivity in live cells. J. Am. Chem. Soc.

2012, 134, 9565–9568.

[12] Peng, H. S.; Stich, M. I. J.; Yu, J. B.; Sun, L. N.; Fischer, L.

H.; Wolfbeis, O. S. Luminescent europium(III)

nanoparticles for sensing and imaging of temperature in the

physiological range. Adv. Mater. 2010, 22, 716–719.

[13] Fischer, L. H.; Harms, G. S.; Wolfbeis, O. S. Upconverting

nanoparticles for nanoscale thermometry. Angew. Chem.,

Int. Ed. 2011, 50, 4546–4551.

[14] Gota, C.; Okabe, K.; Funatsu, T.; Harada, Y.; Uchiyama, S.

Hydrophilic fluorescent nanogel thermometer for

intracellular thermometry. J. Am. Chem. Soc. 2009, 131,

2766–2767.

[15] Jadzinsky, P. D.; Calero, G.; Ackerson, C. J.; Bushnell, D.

A.; Kornberg, R. D. Structure of a thiol

monolayer-protected gold nanoparticle at 1.1 Å resolution.

Science, 2007, 318, 430–433.

[16] Chen, Y.; Zhou, H.; Wang, Y.; Li, W.; Chen, J.; Lin, Q.; Yu,

C. Substrate hydrolysis triggered formation of fluorescent

gold nanoclusters – a new platform for the sensing of

enzyme activity. Chem. Commun. 2013, 49, 9821–9823.

[17] Wang, C. X.; Zhang, D.; Xu, L.; Jiang, Y. N.; Dong, F. X.;

Yang, B.; Yu, K.; Lin, Q. A simple reducing approach using

amine to give dual functional EuSe nanocrystals and

morphological tuning. Angew. Chem. Int. Ed. 2011, 50,

7587–7591.

[18] Dou, X.; Yuan, X.; Yu, Y.; Luo, Z.; Yao, Q.; Leong, D. T.;

Xie, J. Lighting up thiolated Au@Ag nanoclusters via

aggregation-induced emission. Nanoscale 2014, 6,

157–161.

[19] Shang, L.; Dong, S. J.; Nienhaus, G. U. Ultra-small

fluorescent metal nanoclusters: Synthesis and biological


8 Nano Res.

applications. Nano Today, 2011, 6, 401–418.

[20] Liu, J.; Yu, M.; Zhou, C.; Yang, S.; Ning X.; Zheng, J.

Passive tumor targeting of renal-clearable luminescent gold

nanoparticles: Long tumor retention and fast normal tissue

clearance. J. Am. Chem. Soc., 2013, 135, 4978–4981.

[21] Yuan, X.; Setyawati, M. I.; Leong, D. T.; Xie, J. Ultrasmall

Ag+-rich nanoclusters as highly efficient nanoreservoirs for

bacterial killing. Nano Res. 2014, 7, 301–307.

[22] Wang, C.; Huang, Y. Facile preparation of fluorescent

Ag-clusters–chitosan-hybrid nanocomposites for

bio-applications. New J. Chem. 2014, 38, 657–662.

[23] Wang, C.; Wang, C. X.; Xu, L.; Cheng, H.; Lin, Q.; Zhang,

C. Protein-directed synthesis of pH-responsive red

fluorescent copper nanoclusters and their applications in

cellular imaging and catalysis. Nanocale 2014, 6,

1775–1781.

[24] Jia, X. F.; Yuan, X.; Li, J.; Li, D. Y.; Wang, E. Stable Cu

nanoclusters: from an aggregation induced emission

mechanism to biosensing and catalytic applications. Chem.

Commun. 2014, 50, 237–239.

[25] Cauzzi, D.; Pattacini, R.; Delferro, M.; Dini, F.; Natale, C.

D.; Paolesse, R.; Bonacchi, S.; Montalti, M.; Zaccheroni,

N.; Calvaresi, M.; Zerbetto, F.; Prodi, L.

Temperature-dependent fluorescence of Cu5 metal clusters:

A molecular thermometer. Angew. Chem., Int. Ed. 2012, 51,

9662–9665.

[26] Vilar-Vidal, N.; Blanco, M. C.; López-Quintela, M. A.;

Rivas, J.; Serra, C. Electrochemical synthesis of very stable

photoluminescent copper clusters. J. Phys. Chem. C 2010,

114, 15924–15930.

[27] Yuan, X.; Luo, Z.; Zhang, Q.; Zhang, X.; Zheng, Y.; Lee, J.

Y.; Xie, J. Synthesis of highly fluorescent metal (Ag, Au, Pt,

and Cu) nanoclusters by electrostatically induced reversible

phase transfer. ACS Nano 2011, 5, 8800–8808.

[28] Choi, M.; Wu, Z.; Iglesia, E. Mercaptosilane-assisted

synthesis of metal clusters within zeolites and catalytic

consequences of encapsulation. J. Am. Chem. Soc. 2010,

132, 9129–9137.

[29] Zhang; H.; Huang, X.; Li, L.; Zhang, G.; Hussain, I.; Li, Z.;

Tan, B. Photoreductive synthesis of water-soluble

fluorescent metal nanoclusters. Chem. Commun. 2012, 48,

567–569.

[30] Zhang, C.; Zhou, Z.; Qian, Q. ; Gao, G.; Li, C.; Feng, L.;

Wang, Q.; Cui, D. Glutathione-capped fluorescent gold

nanoclusters for dual-modal fluorescence/X-ray computed

tomography imaging. J. Mater. Chem. B 2013, 1,

5045–5053.

[31] Wang, C.; Huang, Y. Green route to prepare biocompatible

and near infrared thiolate-protected copper nanoclusters for

cellular imaging. Nano, 2013, 8, 1350054–1350063.

[32] Kumar, S.; Bolan, M. D.; Bigioni, T. P.

Glutathione-stabilized magic-number silver clusterc. J. Am.

Chem. Soc. 2010, 132, 13141–13143.

[33] Yuan, X.; Zhang, B.; Luo, Z.; Yao, Q.; David, T. L.; Yan, N.;

Xie, J. Balancing the rate of cluster growth and etching for

gram-scale synthesis of thiolate-protected Au25

nanoclusters with atomic precision. Angew. Chem. Int. Ed.

2014, 53, 4623–4627.

[34] Luo, Z.; Zheng K.; Xie, J. Engineering ultrasmall

water-soluble gold and silver nanoclusters for biomedical

applications. Chem. Comm. 2014, 50, 51435155.

[35] Chen, T.; Hu, Y.; Cen, Y.; Chu, X.; Lu, Y. A dual-emission

fluorescent nanocomplex of gold-cluster-decorated silica

particles for live cell imaging of highly reactive oxygen

species. J. Am. Chem. Soc. 2013, 135, 11595–11602.

[36] Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin,

M.; Vezmar, I.; Whetten, R. L. Critical sizes in the growth

of Au clusters. Chem. Phys. Lett. 1997, 266, 9198.

[37] Luo, Z.; Yuan, X.; Yu, Y.; Zhang, Q.; Leong, D. T.; Lee, J.

Y.; Xie, J. From aggregation-induced emission of

Au(I)−thiolate complexes to ultrabright

Au(0)@Au(I)−thiolate core−shell nanoclusters. J. Am.

Chem. Soc. 2012, 134, 1666216670.

[38] Chen, T. H.; Tseng, W. L. (Lysozyme type VI)-stabilized

Au8 clusters: Synthesis mechanism and application for

sensing of glutathione in a single drop of blood. Small 2012,

8, 1912–1919.

[39] Shichibu, Y.; Negishi, Y.; Tsunoyama, H.; Kanehara, M.;

Teranishi, T.; Tsukuda, T. Extremely high stability of

glutathionate-protected Au25 clusters against core etching.

Small 2007, 3, 835839.

[40] Wang, C. X.; Xu, L.; Wang, Y.; Zhang, D.; Shi, X.; Dong,

F.; Yu, K.; Lin, Q.; Yang, B. Fluorescent silver nanoclusters

as effective probes for highly selective detection of

mercury(II) at parts-per-billion levels. Chem. Asian J. 2012,

7, 1652–1656.

[41] Biswas, S.; Miller, J. T.; Li, Y.; Nandakumar, K.; Kumar, C.

S. S. R. Developing a millifluidic platform for the synthesis

of ultrasmall nanoclusters: Ultrasmall copper nanoclusters

as a case study. Small 2012, 8, 688–698.

[42] Zhao, M.; Sun, L.; Crooks, R. M. Preparation of Cu

nanoclusters within dendrimer templates. J. Am. Chem. Soc.

1998, 120, 4877–4878.

[43] Brege, J. J.; Hamilton, C. E.; Crouse, C. A.; Barron, A. R.

Ultrasmall copper nanoparticles from a hydrophobically

immobilized surfactant template. Nano Lett. 2009, 9,

2239–2242.

[44] Goswami, N.; Giri, A.; Bootharaju, M. S.; Xavier, P. L.;

Pradeep, T.; Pal, S. K. Copper quantum clusters in protein

matrix: Potential sensor of Pb2+ Ion. Anal. Chem. 2011, 83,

9676–9680.

[45] Wei, W.; Lu, Y.; Chen, W.; Chen, S. One-Pot Synthesis,

photoluminescence, and electrocatalytic properties of

subnanometer-sized copper clusters. J. Am. Chem. Soc.

2011,133, 2060–2063.

[46] Luo, Z.; Nachammai, V.; Zhang, B.; Yan, N.; Leong, D. T.;

Jiang, D.; Xie, J. Toward understanding the growth

mechanism: Tracing all stable intermediate species from

reduction of Au(I)−thiolate complexes to evolution of Au25

nanoclusters. J. Am. Chem. Soc. 2014, 136, 1057710580.

[47] Wang, C. X.; Wang, Y.; Xu, L.; Shi, X.; Li, X.; Xu, X.; Sun,

H.; Yang, B.; Lin, Q. A galvanic replacement route to

prepare strongly fluorescent and highly stable gold

nanodots for cellular imaging. Small 2013, 9, 413–420.

[48] Yu, Y.; Luo, Z.; Chevrier, D. M.; Leong, D. T.; Zhang, P.;

Jiang, D.; Xie, J. Identification of a highly luminescent

Au22(SG)18 nanocluster. J. Am. Chem. Soc. 2014, 136,

12461249.

[49] Zheng, K.; Yuan, X.; Goswami, N.; Zhang, Q.; Xie, J.

Recent advances in the synthesis, characterization, and

biomedical applications of ultrasmall thiolated silver

nanoclusters. RSC Advances 2014, 4, 6058160596.

[50] Cao, Z. X.; Wang, Y. J.; Zhu, J.; Wu, W.; Zhang, Q. N.

Static polarizabilities of copper cluster monocarbonyls

CunCO (n = 2−13) and selectivity of CO adsorption on


9 Nano Res.

copper clusters. J. Phys. Chem. B 2002, 106, 9649–9654.

[51] Poater, A.; Duran, M.; Jaque, P.; Toro-Labbe, A.; Sola, M.

Molecular structure and bonding of copper cluster

monocarbonyls CunCO (n = 1−9). J. Phys. Chem. B 2006,

110, 6526–6536.

[52] Jia, X.; Li, J.; Wang, E. Cu Nanoclusters with aggregation

induced emission enhancement. Small 2013, 9, 3873–3879.

[53] Xia, F.; Feng, L.; Wang, S. T.; Sun, T. L.; Song, W. L.;

Jiang, W. H.; Jiang, L. Dual-responsive surfaces that switch

between superhydrophilicity and superhydrophobicity. Adv.

Mater. 2006, 18, 432–436.

[54] Shibu, E. S.; Pradeep, T. Quantum clusters in cavities:

Trapped Au15 in cyclodextrins. Chem. Mater. 2011, 23,

989–999.

[55] Wang, S.; Westcott, S.; Chen, W. Nanoparticle

luminescence thermometry. J. Phys. Chem. B 2002, 106,

11203–11209.

[56] Joly, G.; Chen, W.; Roark, J.; Zhang, J. Z. Temperature

dependence of up-conversion luminescence and

photoluminescence of Mn2+ in ZnS: Mn2+ nanoparticles. J.

Nanosci. Nanotech. 2001, 1, 295–301.

[57] Liu, X. J.; Zong, C. H.; Lu, L. H. Fluorescent silver

nanoclusters for user-friendly detection of Cu2+ on a paper

platform. Analyst 2012, 137, 2406–2414.


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.


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.


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.


Electronic Supplementary Material





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.


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].



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.


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.


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.

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