A highly selective fluorescent probe for Cu2+ based on rhodamine B derivative

Article abstract of DOI:10.1016/j.saa.2014.01.046

A new fluorescent probe 1 for Cu²⁺ based on a rhodamine B derivative was designed and synthesized. Probe 1 displays high sensitivity toward Cu²⁺ and about a 37-fold increase in fluorescence emission intensity is observed upon the add

Full text of DOI:10.1016/j.saa.2014.01.046

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A highly selective fluorescent probe for Cu2+ based on rhodamine
B derivative
b,
b
b
c,
Junhong Xu a,b, Yimin Hou b, Qiujuan Ma b, , Xuefen Wu , Suxiang Feng , Juan Zhang , Youming Shen
⇑
⇑
⇑
a Department of Dynamical Engineering, North China University of Water Resources and Electric Power, Zhengzhou 450011, China
b School of Pharmacology, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China
c Department of Chemistry and Chemical Engineering, Hunan University of Arts and Science, Hunan Changde 415000, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
A new rhodamine-based fluorescent
probe for Cu2+ has been developed.
Upon the addition of Cu2+, the probe
exhibits fluorescence enhancement.
The probe shows a high sensitivity
and selectivity for Cu2+
.
Response of probe to Cu2+ is
reversible rather than a cation-
catalyzed reaction.
ꢀ
The probe has been used for imaging
of Cu2+ in living cells.
a r t i c l e i n f o
a b s t r a c t
A new fluorescent probe 1 for Cu2+ based on a rhodamine B derivative was designed and synthesized.
Probe 1 displays high sensitivity toward Cu2+ and about a 37-fold increase in fluorescence emission inten-
sity is observed upon the addition of 10 equiv. Cu2+ in 50% water/ethanol buffered at pH 7.10. Besides,
upon binding Cu2+ a remarkable color change from colorless to pink was easily observed by the naked
eyes. The reversible dual chromo- and fluorogenic response toward Cu2+ is likely due to the chelation-
induced ring-opening of rhodamine spirolactam. The linear response range covers a concentration range
of Cu2+ from 8.0 ꢁ 10ꢂ7 to 1.0 ꢁ 10ꢂ4 mol/L and the detection limit is 3.0 ꢁ 10ꢂ7 mol/L. Except Co2+, the
probe exhibits high selectivity for Cu2+ over a large number of cations such as alkaline, alkaline earth and
transitional metal ions. The accuracy and precision of the method were evaluated by the analysis of the
standard reference material, copper in water (1.0 mol/L HNO3). The proposed probe has been used for
direct measurement of Cu2+ content in river water samples and imaging of Cu2+ in living cells with sat-
isfying results, which further demonstrates its value of practical applications in environmental and bio-
logical systems.
Article history:
Received 24 September 2013
Received in revised form 16 December 2013
Accepted 10 January 2014
Available online 22 January 2014
Keywords:
Fluorescent probe
Copper ion
Rhodamine B derivative
Salicylaldehyde
Spirolactam
Fluorescence enhancement
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
and plays a pivotal role in various fundamental physiological pro-
cesses in organisms ranging from bacteria to mammals [1]. How-
Copper is a transition element essential to plants and animals,
including humans. After Fe2+ and Zn2+, Cu2+ ranks the third in
abundance among the essential trace elements in the human body,
ever, it is also harmful to biological systems in excessive
amounts. Alteration in the cellular homeostasis of copper ions
are associated with neurodegenerative disease, including Menkes
and Wilson diseases, familial amyotropic lateral sclerosis, Alzhei-
mer’s disease, an prion diseases [2]. The Word Health Organisation
(WHO) has set the maximum allowable level of copper in drinking
⇑
Corresponding authors. Tel.: +86 371 65676656; fax: +86 371 65680028.
E-mail addresses: maqiujuan104@126.com (Q. Ma), wxf568@163.com (X. Wu).
1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2014.01.046

J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
417
water at 2 mg/L (31
l
M) [3]. Nevertheless, copper contamination
tilled water was used throughout all experiments. Thin layer chro-
matography (TLC) was carried out using silica gel 60 F254, and
column chromatography was conducted over silica gel (200–300
mesh), both of which were obtained from the Qingdao Ocean
Chemicals (Qingdao, China). The Cu standard solution, copper in
water (1.0 mol/L HNO3) (GSB 04-1725-2004) was obtained from
Analysis and Testing Center of National Nonferrous Metals and
Electronic Materials (Beijing, China).
and its potential toxic effects on biological systems are still the
challenging problems throughout the world because of copper
wide use for many industrial, agricultural and domestic purposes.
Accordingly, it is very important to monitor copper levels in vari-
ous samples for environment and human health.
Thus far, various efficient and reproducible methods have been
proposed to detect the concentrations of Cu2+ including atomic
absorption spectrometry (AAS) [4], inductively coupled plasma–
atomic emission spectrometry (ICP–AES) [5], inductively coupled
plasma–mass spectrometry (ICP–MS) [6], etc. Though these tech-
niques are sensitive, selective, and accurate for Cu2+ assay, they
are usually complicated, time-consuming, and costly. Due to their
advantages of high sensitivity, specificity, and ease of operation, a
large number of fluorescent probes have been reported for the
determination of copper ions so far. However, most of them exhibit
fluorescence emission quenching upon binding Cu2+ owing to the
paramagnetic nature of Cu2+ [7–10]. Fluorescence quenching is
not only unfavourable for a high signal output upon recognition
but also hinders temporal separation of spectrally similar com-
plexes with time-resolved fluorometry [11]. Until now, only a
few probes with a Cu2+ induced fluorescence enhancement have
been proposed [2,12,13]. Thus, searching for Cu2+ probes based
on fluorescence enhancement is still an active field as well as a
challenge for the analytical chemistry research effort.
Rhodamine and its derivatives are excellent fluorophores and
chromophores owing to their very good spectroscopic properties,
such as large molar extinction coefficient, relatively long excitation
and emission wavelengths elongated to visible region, high light
stability, and high fluorescence quantum yield [14]. Rhodamine
derivatives with a spirolactam-ring moiety are non-fluorescent
and colorless. In the presence of a proton or metal cation, they
can be converted to the open-ring forms via a reversible coordina-
tion or an irreversible chemical reaction and give rise to strong
fluorescence emission and a pink color [15]. Thus, the rhodamine
framework is an ideal model to fabricate the turn-on fluorescent
probe. In the past few years, several rhodamine-modified probes
have been developed for various cations such as Cu2+ [16–18],
Hg2+ [19–21], Fe3+ [22–24], Pb2+ [25], Cr3+ [26], Ag+ [27], Zn2+
[28], and Cr5+ [29]. According to the Soft–Hard Acid–Base theory,
the probes attached the recognition moiety with N and O atoms
could display good affinity to Cu2+. Recently, several probes based
on rhodamine and salicylaldehyde derivatives have been widely
used for detecting Cu2+ [30–39]. However, these probes contain
hydrazone group which makes them unstable for the long term
storage neither in the air nor in the solvent [40]. Herein, a Cu2+
fluorescent probe (probe 1) based on the rhodamine hydrazone
derivative has been developed. It exhibits fluorescence enhance-
ment upon the addition of Cu2+ in 50% water/ethanol buffered at
pH 7.10 and its high selectivity for Cu2+ in the presence of many
other metal cations. Furthermore, the proposed probe has been
used for direct measurement of Cu2+ content in river water sam-
ples and imaging of Cu2+ in living cells with satisfying results.
Syntheses
The synthetic route for fluorescence probe 1 was shown in
Scheme 1.
Compound 2 was synthesized according to Xiang’s method [16]
by the reaction of rhodamine B and hydrazine hydrate (85%). Com-
pound 3 was prepared starting from compound 2 and salicylalde-
hyde by the reported literature [16].
Synthesis of compound 1
To a stirred solution of compound 3 (0.50 g, 1 mmol) in anhy-
drous CH2Cl2 (20 mL) cooled to 0 °C was carefully added solid
sodium borohydride (0.19 g, 5 mmol) in portions. The resulting
mixture was stirred at room temperature for 48 h. After reaction,
the reaction mixture was filtrated and the filtrate was evaporated
under reduced pressure. The crude product was purified by silica
gel column chromatography using petroleum ether/CH3COOCH2
CH3 (7:1, V/V) as eluent to afford compound 1 (0.23 g, 40%) as a
white solid. 1H NMR (400 MHz, CDCl3), d(ppm): 1.16 (t, 12H, NCH2
CH3, J = 7.2 Hz), 3.33 (q, 8H, NCH2CH3, J = 7.2 Hz), 3.86 (d, 2H,
NHCH2, J = 6.8 Hz), 4.22 (t, 1H, NHCH2, J = 6.8 Hz), 6.24 (dd, 2H,
Xanthene-H, J = 8.8 Hz, 2.8 Hz), 6.43 (m, 2H, Xanthene-H), 6.45
(m, 2H, Xanthene-H), 6.68 (m, 1H, Phen-H), 6.81 (d, 1H, Phen-H,
J = 8.0 Hz), 6.85 (dd, 1H, Phen-H, J = 7.4 Hz, 1.6 Hz), 7.10 (m, 1H,
Phen-H), 7.14 (dd, 1H, Ar-H, J = 7.2 Hz, 1.6 Hz), 7.50 (m, 2H, Ar-
H), 7.95 (m, 1H, Ar-H), 8.80 (bs, 1H, Phen-OH).
Apparatus
All fluorescence measurements were made on a Hitachi F-4500
Fluorescence Spectrometer (Tokyo, Japan) in 1 cm ꢁ 1 cm quartz
cell. UV–Vis absorption spectra were recorded on a Shimadzu
UV-2450 UV–Vis spectrophotometer (Tokyo, Japan) in 1 cm path
length quartz cuvettes with a volume of 4 mL. 1H NMR was ac-
quired in CDCl3 on Varian INOVA-400 MHz NMR spectrometer
using TMS as an internal standard. The measurements of pH were
carried out on a Mettler-Toledo Delta 320 pH meter (Shanghai, Chi-
na) with a Mettler combination glass electrode (No. 4140230002).
The electrode was calibrated for pH using commercial pH reference
solutions (pH 4.00, pH 6.86 and pH 9.18 standard solutions). Data
processing was performed on an Inter Core i5 computer with soft-
ware of SigmaPlot.
Measurement procedures
A stock solution of 2.0 ꢁ 10ꢂ5 mol/L compound 1 was obtained
by dissolving the requisite amount of 1 in absolute ethanol. A stan-
dard stock solution of 1.0 ꢁ 10ꢂ2 mol/L Cu2+ was prepared by dis-
solving an appropriate amount of copper chloride in water and
adjusting the volume to 100 mL in a volumetric flask. Working
solutions of copper chloride were freshly prepared by serial dilu-
tion of the stock solution with 0.05 M Tris–HCl buffer solution.
The complex solution of Cu2+ and compound 1 was obtained by
mixing 12.5 mL of the stock solution of compound 1 and 2.5 mL
of Cu2+ solution of the different concentrations in a 25 mL volumet-
ric flask. Then the mixture was diluted to 25 mL with Tris–HCl buf-
fer solution. In the solution thus obtained, the concentrations were
Experimental
Reagents
Rhodamine B, hydrazine hydrate (85%), salicylaldehyde, sodium
borohydride and ethylenediaminetetraacetic acid disodium salt
(EDTA-2Na) were obtained from Shanghai Chemical Reagents and
used as received. Before being used, dichloromethane was distilled
at atmospheric pressure from CaH2 and stored over 4 Å molecular
sieves. Other chemicals were of analytical reagent grade and used
without further purification except when specified. Doubly dis-

418
J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
HO
O
O
O
NH2
N
N
b
N
OH
a
N
O
N
N
O
N
N
O
N
Cl
3
2
HO
O
H
N
N
c
N
O
N
1
Scheme 1. Synthesis of fluorescent probe 1. (a) hydrazine hydrate(85%), ethanol, reflux, 12 h, 73%; (b) salicylaldehyde, ethanol, reflux, 5 h, 55%; (c) NaBH4, anhydrous CH2Cl2,
48 h, 40%.
1.0 ꢁ 10ꢂ5 mol/L in compound 1 and 8.0 ꢁ 10ꢂ7–1.0 ꢁ 10ꢂ3 mol/L
in Cu2+. Blank solution of compound 1 was prepared under the
same conditions without Cu2+. The salts used in other stock solu-
tions of metal ions were LiCl, NaCl, KCl, MgSO4, AlCl3, CaCl2, MnCl2,
AgNO3, Pb(NO3)2, Hg(NO3)2ꢃ0.5H2O, (CH3CO2)2Cdꢃ2H2O, Co(CH3
CO2)2ꢃH2O, NiSO4ꢃ6H2O, ZnSO47H2O, Fe2(SO4)3. The wide pH range
solutions were prepared by adjustment of 0.05 mol/L Tris–HCl
solution with HCl or NaOH solution. For all measurements of fluo-
rescence spectra, excitation was fixed at 520 nm with excitation
slit set at 10.0 nm and emission at 10.0 nm. The voltage of the pho-
tomultiplier tube (PMT) detector is 700 V.
Cell incubations
H292 lung cancer cells were cultured in RPMI Medium 1640
(Gibco) containing 10% calf bovine serum (HyClone) at 37 °C in
humidified air and 5% CO2. H292 lung cancer cells grown on cover-
Fig. 1. Changes of the fluorescence spectra of probe 1 (10
various concentrations of Cu2+: 0, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 20, 30, 40, 60, 80,
100 M from 1 to 14 (kex = 520 nm). These spectra were measured in 0.05 M Tris–
lM) in the prescence of
slips were washed with phosphate-buffered saline (PBS), followed
by incubating with 10 M of probe 1 in DMSO–RPMI Medium 1640
(1:99, V/V) for 30 min at 37 °C, and then washed with PBS three
times. After incubating with 10 M of CuCl2 in PBS for 1 h at
l
l
HCl buffer (ethanol/water = 1:1, v/v, pH 7.10).
l
37 °C, the cells were washed with PBS three times again.
In order to obtain a better insight into the response mechanism
of 1 toward Cu2+, the absorption spectra of 1 in the absence and
presence of Cu2+ were recorded (Fig. 2). The colorless free 1
Fluorescence imaging
(10 lM) in ethanol/water (1:1, v/v) exhibited almost no absorption
Confocal fluorescence imaging was performed with a Zeiss LSM
700 laser scanning microscope with 20ꢁ objective lens. Excitation
of 1-loaded cells at 555 nm was carried out with a solid laser and
emission was collected at 560–1000 nm.
near 560 nm, which could be ascribed to the closed spirolactam
form of probe 1 in the absence of Cu2+. Upon addition of
1.0 ꢁ 10ꢂ3 M Cu2+ to the solution of probe 1, a new absorption
peak at 559 nm (
e
= 1.20 ꢁ 103 L molꢂ1 cmꢂ1) emerged and the
solution displayed a clear change from colorless to pink, indicating
the formation of the ring-opened amide form of probe 1 in the
presence of Cu2+ [15]. Job’s method for the absorbance was applied
to determine the stoichiometry between 1 and Cu2+, by keeping the
sum of the initial concentration of copper ion and at 1.0 ꢁ 10ꢂ4
mol/L (Fig. 3) [41]. As depicted in Fig. 3, a maximum absorbance
was showed when the molecular fraction of Cu2+ was close to
0.5, which indicated the 1:1 complex formation between 1 and
Cu2+. Thus, a possible coordination mode for probe 1 with Cu2+
was proposed (Scheme 2).
Results and discussion
Spectral characteristics
Fig. 1 exhibits fluorescence emission changes of 10 lM probe 1
in buffered (Tris–HCl, pH = 7.10) ethanol/water (1:1, v/v) upon
addition of different Cu2+ concentrations. As can be seen from
Fig. 1, free 1 shows very slight fluorescence in the 540–650 nm
range and the fluorescence intensities of probe 1 increase obvi-
ously at 576 nm with increasing concentration of Cu2+. When the
concentration of Cu2+ is up to 10 equiv. of compound 1, an approx-
imately 37-fold enhancement in the fluorescence emission inten-
sity was observed. These results constitute the basis for the
determination of Cu2+ concentration with probe 1 proposed in this
work.
Principle of operation
On the basis of 1:1 stoichiometric relationship mentioned
above, the linear response of the fluorescence emission intensity

J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
419
2þ
Fig. 4. Plot of fluorescence intensity of the proposed probe 1 as a function of C
.
Cu
Fig. 2. UV-spectra of probe 1 (10
l
M) before (—) and after (ꢃꢃꢃ) the addition of Cu2+
These data were measured in pH 7.10 Tris–HCl buffer (ethanol/water = 1:1, v/v).
The excitation wavelength was 520 nm. Measurements were carried out in
triplicate.
(1.0 ꢁ 10ꢂ3 mol/L) in pH 7.10 tris–HCl buffer (ethanol/water, 1:1, v/v).
10 measurements of the blank and m is the slope of the calibration
line) was 3.0 ꢁ 10ꢂ7 mol/L [42]. Thus, our proposed probe 1 could
sensitively detect environmentally relevant levels of Cu2+
.
Effect of pH
To study the practical applicability, the effects of pH on the fluo-
rescence intensity of probe 1 in the absence Cu2+ were investigated
in the pH range from 3.00 to 13.00 (Fig. 5). As shown in Fig. 5,
probe 1 did not show any obvious and characteristic fluorescence
(excitation at 520 nm) in the pH range of 6.00–12.00, suggesting
that it was insensitive to pH and that the spirolactam form is still
preferred in this condition. When pH was lower than 6.00, the fluo-
rescence intensity increased with decreasing pH values, which
might be caused by the ring-opened form of 1 in the presence of
proton. From the view of sensitivity and the speed time, the
Tris–HCl buffer solution at pH 7.10 (ethanol/water, 1:1, v/v) was
used as an ideal experimental media.
Fig. 3. Job’s plot for probe 1 in 0.05 M tris–HCl buffer (ethanol/water = 1:1, v/v, pH
7.10). The total concentration of 1 and Cu2+ was 100
lM.
Selectivity
H
O
H
O
Selectivity is a very important parameter to evaluate the perfor-
mance of a fluorescence probe. The selectivity experiments were
carried out by fixing the concentration of ion at 1.0 ꢁ 10ꢂ4 mol/L,
Cu2+
N
N
N
O
N
Scheme 2. Possible coordination mode for probe 1 with Cu2+
.
of probe 1 toward amounts of Cu2+ added was obtained in Cu2+
concentration range of 8.0 ꢁ 10ꢂ7 to 1.0 ꢁ 10ꢂ4 mol Lꢂ1 (Fig. 4).
The linear response can be expressed by the following Eq. (1) of
the calibration line:
F ¼ 113:3242 þ 27:0467 ꢁ 106C2þ ðR ¼ 0:9979Þ
ð1Þ
Cu
Here F is the fluorescence emission intensity of probe 1 actually
2þ
measured at a given metal concentration, and C represents the
Cu
Fig. 5. pH dependence of the fluorescence intensity of 10 lM probe 1 in the absence
concentration of Cu2+ added. The detection limit attained for
of Cu2+. All data were obtained at various pH values (pH 3.00–13.00) and the
excitation wavelength was 520 nm.
Cu2+, calculated by 3 sB/m (where sB is the standard deviation of

420
J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
and various cations were added as chlorides, nitrates, acetate and
sulfates. Fig. 6 (the black bar portion) illustrated the fluorescence
responses of probe 1 to various cations. As can be seen from the
black bars in Fig. 6, only Co2+ cause a mild fluorescence intensity
enhancement, while the other metal ions not induce any obvious
fluorescence interferences, indicating that our proposed probe
exhibits high selectivity for the Cu2+ ion over other metal ions.
To test practical applicability of our fluorescent probe for Cu2+
,
competition experiments were performed in which the fluorescent
probe was added to a solution of Cu2+ in the presence of other me-
tal ions (white bars in Fig. 6). Experimental results indicate that the
response of the probe to Cu2+ is unaffected by the presence of the
other possible contaminating metal ions. It can be found that ex-
cept Co2+ the relative error of common interference such as alkali,
alkaline earth and transitional metal ions was less than 5%, which
is considered as tolerated. Fortunately, Co2+ could be effectively
circumvented by adding masking reagent Fꢂ. All these selective re-
sults indicate that our proposed probe could meet the selective
requirements for practical applications.
Fig. 7. Reversible fluorescence response of 1 to Cu2+ in pH 7.10 Tris–HCl buffer
(ethanol/water, 1:1, v/v). —: 10 M probe 1; ----: 10 M probe 1 with 100
M Cu2+
ꢃ ꢃ ꢃ: 10 M probe 1 with 100 M EDTA-2Na.
M Cu2+ and then addition of 400
Excitation was performed at 520 nm.
l
l
l
l
;
l
l
Reversibility and long-term stability
water samples obtained from different locations of Jinshui River,
Zhengzhou and simply filtrated. In order to reduce pH influence
on the Cu2+ detection, the pH values of sample solutions were buf-
fered to pH 7.10. The results were summarized in Table 1, which
agreed well with the atomic absorption spectrometry reference
method with a relative deviation less than 3%. These results dem-
onstrated that our proposed fluorescent probe could meet the sen-
sitivity as well as selectivity requirements for environmental water
samples monitoring applications. In order to evaluate the accuracy
using the proposed method, the determination of copper in stan-
dard reference materials, copper in water (1.0 mol/L HNO3) (GSB
04-1725-2004) was carried out. The copper mass concentration
in the Cu standard solution (GSB 04-1725-2004) was referenced
As is well known, the reversibility is important for the fabrica-
tion of devices to sense copper ion. Because of the high stability
constant of the EDTA-Cu, the EDTA-adding experiments were con-
ducted to examine the reversibility of the probe 1 in Fig. 7. Firstly,
the addition of Cu2+ to the free 1 solution makes the fluorescence
increase significantly. Secondly, the pink color and fluorescence
of the solution containing 1 and Cu2+ disappeared instantly upon
the addition of excess EDTA. The above results elicit that the spec-
tral response of probe 1 to Cu2+ is reversible rather than a cation-
catalyzed reaction [43].
In order to examine the long-term stability of the prepared
probe, a stock solution of 2.0 ꢁ 10ꢂ5 mol/L compound 1 in absolute
ethanol and a standard stock solution of 1.0 ꢁ 10ꢂ2 mol/L Cu2+ in
water were stored in a refrigerator when not in use. The probe
works well and no detectable change in working concentration
range is found for one month, which implies that the proposed
probe is stable in absolute ethanol.
to be 1000
lg/mL. The measured copper mass concentration in
the Cu standard solution (GSB 04-1725-2004) was 999.5 7.9
lg/
mL, which was in good agreement with the certified value. Thus,
the proposed sensor seems useful for the determination of iron
ions in real samples.
To further demonstrate the practical applicability of the pro-
posed probe in biological samples, fluorescence imaging experi-
ments were carried out in living cells (Fig. 8). Incubation of H292
Preliminary analytical application
lung cancer cells with 10
weak fluorescence (Fig. 8A). When H292 lung cancer cells were incu-
bated with 10
M Cu2+, the same treatment with probe 1 generated
lM of probe 1 for 30 min at 37 °C gave very
In order to test the practical application of the proposed probe,
the probe was first applied in the determination of Cu2+ in the river
l
much brighter fluorescence (Fig. 8D). The results suggested that
probe can penetrate the cell membrane and can be applied for
in vitro imaging of Cu2+ in living cells and potentially in vivo.
Method performance comparison
The performance of the proposed probe toward Cu2+ was com-
pared with some reported fluorescent probes based on either rho-
damine or salicylaldehyde derivative for Cu2+ determination, as
Table 1
Results of Cu2+ determination in river water samples.
Sample
Concentration of copper (M)
Relative
error (%)
The proposed
sensor
Atomic absorption
spectrometry
River water 1 (11.81a 0.24b) ꢁ 10ꢂ6 (11.68a 0.18b) ꢁ 10ꢂ6
1.11
2.55
ꢂ2.13
River water 2 (8.86a 0.13b) ꢁ 10ꢂ6
River water 3 (6.43a 0.07b) ꢁ 10ꢂ6
(8.64a 0.15b) ꢁ 10ꢂ6
(6.57a 0.12b) ꢁ 10ꢂ6
Fig. 6. Metal ion selectivity of probe 1 (10 lM). All data were obtained at pH 7.10
Tris–HCl buffer (ethanol/water = 1:1, v/v). The concentration of ions added to probe
1 was 1.0 ꢁ 10ꢂ4 mol/L for all metal ions. The excitation wavelength was 520 nm.
Black bars: different metal ions were added. White bars: different metal ions in the
presence of Cu2+ were added.
a
Mean value of the three determinations.
Standard deviation.
b

J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
421
Fig. 8. Images of H292 lung cancer cells treated with the presented probe 1. (A) Fluorescence image of H292 lung cancer cells incubated with 1 (10
lM) for 30 min. (B) Bright-
field transmission image of cells shown in panel (A). (C) Overlay image of (A) and (B). (D) Fluorescence image of H292 lung cancer cells incubated with 1 (10
l
M) for 30 min,
washed three times, and then further incubated with 10 l
M Cu2+ for 1 h. (E) Bright-field transmission image of cells shown in panel (D). (F) Overlay image of (D) and (E).
Table 2
The comparison of the proposed probe with some other fluorescence probes for the measurement of Cu2+
.
Modes
Reagents
Linear range (M)
Limit of
detection
(M)
Working media
Interference Applications
of other
metal ions
Refs.
[30]
Quenching kex/em = 529/575 nm
The mixture of Rhodamine 1 ꢁ 10ꢂ7–3.6 ꢁ 10ꢂ6 2.5 ꢁ 10ꢂ8
pH 7.0 buffer EtOH/
H2O (1:1, V/V)
solution
No
Water samples
B and rhodamine
derivative
Enhancement kex/em = 440/510 nm Salicylaldehyde derivative 2 ꢁ 10ꢂ6ꢂ4.8 ꢁ 10ꢂ6 4.477 ꢁ 10ꢂ7 pH 7.0 buffer EtOH/
Co2+, Hg2+ Fluorescent imaging [39]
in Hela cells
H2O (9:1, V/V)
solution
CH2Cl2
Quenching kex/em = 365/512 nm
Salicylaldehyde derivative Not discussed
6 ꢁ 10ꢂ7
Co2+
Hg2+
Molecular logic
switch
No
[44]
[45]
[46]
Enhancement kex/em = 480/530 nm Rhodamine
Not discussed
Not discussed CH3CN
derivative
Rhodamine
derivative
Enhancement kex/em = 552/
580 nm
2 ꢁ 10ꢂ6–2.0 ꢁ 10ꢂ5 2 ꢁ 10ꢂ6
pH 7.0 buffer MeOH/ Zn2+
H2O (1:1, V/V)
No
solution
pH 7.4 buffer MeOH/ No
H2O (1:1, V/V)
solution
pH 7.04 buffer CH3CN/ No
H2O (2:8, V/V)
Enhancement kex/em = 550/
576 nm
Rhodamine
derivative
Not discussed
Not discussed
3.0 ꢁ 10ꢂ6
2.0 ꢁ 10ꢂ7
3.0 ꢁ 10ꢂ7
Fluorescent imaging [47]
in Hela cells
Enhancement kex/em = 530/
571 nm
Rhodamine
derivative
No
[41]
solution
Enhancement kex/em = 520/
576 nm
Rhodamine
derivative
8.0 ꢁ 10ꢂ7
1.0 ꢁ 10ꢂ4
–
pH 7.10 buffer EtOH/ Co2+
H2O (1:1, V/V)
Fluorescent imaging This
in H292 lung cancer work
cells and water
solution
samples
shown in Table 2. It can be found that reported fluorescence probe
for Cu2+ usually have interference problems caused by other tran-
sition metals cations such as Hg2+, Zn2+ and Co2+ [39,44–46]. But
the proposed sensor in this work exhibits good selectivity and
has no interference of other transition metals cations except
Co2+. Moreover, the proposed probe in this paper displays wide lin-
ear span and low detection limit compared with those probes in
Refs. [39,46,47]. The two probes in Refs. [44,45] need more rigor-
ous testing media and the applicability in living cells are not inves-
tigated. The applications in living cells in Refs. [30,44–46,41] are
also not validated. Therefore, from Table 2 it was not difficult to
find that our newly developed sensor for detecting of Cu2+ featured
with high selectivity, a wide linear range and wide applicability.
Conclusion
In summary, we have designed and synthesized a new rhoda-
mine-based fluorescent probe for copper ions. Our proposed probe
displays a reversible absorption and fluorescence enhancement
response to Cu2+ via a 1:1 binding mode. Moreover, probe 1 shows

422
J. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 416–422
a high sensitivity and selectivity for Cu2+ sensing in comparison to
other cations in 50% water/ethanol buffered at pH 7.10. It could
work over a wide pH range from 6.00 to 13.00, which is important
for possible use in practical view to selective requirements for bio-
medical and environmental application. The proposed probe can be
applied to the quantification of Cu2+ with a linear range covering
from 8.0 ꢁ 10ꢂ7 to 1.0 ꢁ 10ꢂ4 mol/L and the detection limit is
3.0 ꢁ 10ꢂ7 mol/L. All of its merits make it adequate for practical
application of Cu2+ array. The present probe has been used for di-
rect measurement of Cu2+ content in river water samples and
imaging of Cu2+ in living cells with satisfying results, which further
demonstrates its value of practical applications in environmental
and biological systems.
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This work was supported by High-level Talent Projection for
North China University of Water Resources and Electric Power
(40334), Program for Science & Technology Innovation Talents in
Henan College of Chinese Medcine, Program for Innovative Culture
Research Team (in Science an Technology) in Henan College of Chi-
nese Medcine, Henan Natural Science Foundation of Education
Department (2011B360005 and 14B150021) and the National Nat-
ural Science Foundation of China (21342012).
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