Rhodamine-based derivatives for Cu2+ sensing: Spectroscopic studies, structure-recognition relationships and its test strips

Article abstract of DOI:10.1016/j.saa.2011.05.016

A rhodamine spirolactam/2-hydrazinopyridine derivative was synthesized and characterized, which exhibited high selectivity to Cu²⁺ over other metal cations. The Cu²⁺ recognition of this rhodamine derivative could be detected by fluor

Full text of DOI:10.1016/j.saa.2011.05.016

Spectrochimica Acta Part A 81 (2011) 14–20
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Rhodamine-based derivatives for Cu2+ sensing: Spectroscopic studies,
structure-recognition relationships and its test strips
Yunxu Yanga,∗, Weiling Gaoa, Ruilong Shengb,∗∗, Weili Wanga, Hui Liua, Wumin Yangc,
Tianyi Zhanga, Xuetao Zhanga
a Department of Chemistry and Chemical Engineering, University of Science and Technology Beijing, 100083, China
b Shanghai Institute of Organic chemistry, Chinese Academy of Sciences, Shanghai, 200032, China
c Yixing foreign language school, Wuxi, 210005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A rhodamine spirolactam/2-hydrazinopyridine derivative was synthesized and characterized, which
exhibited high selectivity to Cu2+ over other metal cations. The Cu2+ recognition of this rhodamine
derivative could be detected by fluorescence spectra, absorption spectra and an obvious color change
which was observed easily by naked-eyes. The binding of this rhodamine derivative to Cu2+ is instan-
taneous and sensitive. Moreover, a linear relationship was found between the fluorescence intensity
at 575 nm from 0.5 × 10−6 M to 3.0 × 10−6 M of Cu2+ concentration, and the limit of detection (LOD)
was at low concentration of 2.11 × 10−8 M, this would benefit for the establishment of standard
working curves in practical Cu2+detection. Additionally, we synthesized rhodamine spirolactam/2-
aminomethylpyridine derivative and rhodamine spirolactam/phenylhydrazine derivative as analogs for
elucidate the structure-recognition relationships. Finally, we prepared the test strips of rhodamine
spirolactam/2-hydrazinopyridine derivative for practical chromogenic the Cu2+ detection.
© 2011 Elsevier B.V. All rights reserved.
Received 24 February 2011
Received in revised form 10 May 2011
Accepted 16 May 2011
Keywords:
Spectroscopy
Rhodamine
Cu2+ detection
Structure-recognition relationships
Test strips
1. Introduction
chemosensors were synthesized and employed in aqueous media
[18,19]. So, developing new chemosensors or their derived sensing
The development of increasingly selective and sensitive meth-
ods for the determination of heavy metal ions is currently receiving
considerable attention [1–4]. Among the various detection meth-
ods available, UV–vis and fluorescence spectroscopy still remain
the most frequently used modes for the recognition of physiolog-
ically and environmentally important analytes due to their high
sensitivity and easy operational use.
materials for rapid and convenient Cu2+ detection are still highly
demanded.
Rhodamine-based compounds were found to be good flu-
orescent “Turn-on/off” switches. Since a ring opening-closing
conversion (Scheme 1) between non-fluorescent (colorless) rho-
damine spirolactam form and highly fluorescent (red) rhodamine
ring-opening form could be induced by metal cations [20], based on
the mechanism, many rhodamine derivated fluorescent chemosen-
sors/chemodosimeters had been developed for the detection of
various metal ions [21–28]. Dujols firstly utilized a rhodamine
B hydrazide as a chemodosimeter for Cu2+ sensing in aqueous
solution [29], after that, some metal-chelating moieties bearing
rhodamine derivatives [30,31] were developed as a sort of highly
selective Cu2+ chemodosimeters. Zhao [32] synthesized a Cu2+
selective chemodosimeter by incorporating a thiosemicarbazide
moiety with rhodamine B, Chen [33] reported a chemosensor bear-
ing rhodamine-binaphthyl groups to detect Cu2+, Zeng [34] given
a tripodal rhodamine receptor as Cu2+ and Hg2+ chemosensor, and
Yu’s research group [35] compared several carbohydrazone rho-
damine derivatives for their Cu2+ recognition properties. These
progresses were listed in Table 1. Although number of rhodamine-
based Cu2+ chemosensors had been developed, the sensitivity
and efficiency for Cu2+ detection should be further improved. On
the other hand, how to rationally design a preferred molecular
Cu2+ plays important roles in biological and environmental
processes. The development of Cu2+ sensing materials with high
selectivity and sensitivity is attracting increasing attention in ana-
lytical, biological and environmental sciences. However, most of
the early-reported Cu2+ chemosensors are fluorescence-quenching
types [5–10]. Factors such as the interference of coexisting metal
ions, the convenient portability and the rapidly response still
limited the practical application of the Cu2+ chemosensors. For
the consideration of more sensitive detection, many fluores-
cent “Turn-on” chemosensors have been developed [11–17]; for
the improvement of the water solubility, some water-soluble
∗
Corresponding author. Tel.: +86 10 62333871; fax: +86 01 62332462.
Corresponding author. Tel.: +86 21 54925255.
E-mail addresses: yxyang63@yahoo.com.cn (Y. Yang),
∗∗
rayleigh121@yahoo.com.cn (R. Sheng).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.05.016

Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
15
Scheme 1. Metal cations induced ring opening mechanism for rhodamine-based chemosensors.
structure for Cu2+ sensing is still a challenge. Therefore, design and
synthesis of highly efficient rhodamine-based Cu2+ chemosensors
and elucidate their structure-recognition relationship are desir-
able.
2.2. Synthesis of 1
To a solution of rhodamine B (1.0 g, 2.3 mmol) in dry 1,2-
dichloroethane (8.0 mL), phosphorus oxychloride (1.1 g, 6.9 mmol)
was added dropwise at room temperature within 5 min. After being
refluxed for 4 h, the reaction mixture was cooled and concentrated
under vacuum to give rhodamine B acid chloride. The crude product
was used directly in the next reaction without purification.
The crude rhodamine B acid chloride in last step was dissolved
in dry acetonitrile (10 mL), and added dropwise to a solution of
2-hydrazinopyridine (4.6 mmol, 0.67 g) in dry acetonitrile (6.0 mL)
containing triethylamine (8.0 mL). After stirring for 4 h at room
temperature, the mixture was concentrated under vacuum and the
crude product was purified by column chromatography (eluant:
ethyl acetate/Petroleum ether; v/v = 1/20) to give compound 1 as
a white solid in 70% yield. 1H NMR (400 MHz, CDCl3), ı (ppm):
1.13–1.16 (t, 12H, J = 7.2 Hz, NCH2CH3), 3.29–3.35 (q, 8H, J1 = 6.8 Hz,
J2 = 7.2 Hz, NCH2CH3), 6.24–6.29 (m, 5H, ArH), 6.50–6.57 (m, 3H,
ArH), 7.14–7.18 (t, 1H, J = 7.2 Hz, ArH), 7.26–7.28 (m, 1H, ArH),
7.54–7.63 (m, 2H, ArH), 7.96–7.97 (d, 1H, J = 4.0 Hz, ArH), 8.02–8.04
(d, 1H, J = 8.0 Hz, ArH); MS (ESI) m/z: 534.3 (M + H+, 100%).
In this work, we designed and synthesized a rhodamine
spirolactam/2-hydrazinopyridine derivative 1 as a chemosensor,
which showed highly selectivity and sensitivity for Cu2+ over
other coexistent metal cations in aqueous media. The fluores-
cent “Turn-on” process could be easily detected by means of
spectroscopy or directly observed by naked eyes. Factors such as
concentration dependence, pH effect, response time and coexisted
metal ions that influenced Cu2+ sensing were also investigated
and discussed. Additionally, comparing to most of the researches
reported, which only demonstrated the recognition properties of
the chemosensors themselves, two other analogs of rhodamine
spirolactam/2-aminomethylpyridine derivative 2 and rhodamine
spirolactam/phenyl hydrazine derivative 3 were synthesized in this
work for the investigation of structure-recognition relationships
(Scheme 2). Derivatives 2, 3 showed much less fluorescence sen-
sitivity to Cu2+ than that of 1 and these were benefits for rational
design of more advanced chemosensors. For practical application,
paper-made test strips were successfully prepared for aqueous Cu2+
detection.
2.3. Synthesis of 2
To a 100 mL flask, rhodamine B acid chloride (0.6 g, 1.3 mmol)
was dissolved in 30 mL methanol. Excess of 2-aminomethyl-
pyridine (0.36 g, 3.4 mmol) was then added dropwise with vigorous
stirring at room temperature within 20 min. Then the mixture was
heated to reflux for 24 h. The solution changed from dark purple to
light orange. Then the mixture was cooled down and the solvent
was removed under reduced pressure. 1 M HCl (50 mL) was added
to dissolve the residue, then 1 M NaOH (70 mL) was added slowly
with stirring until the pH of the solution reached pH = 9–10. The
resulting precipitate was filtered and washed 3 times with 15 mL
of water, and then dried in a vacuum drying oven to afford 2 as a
pink solid (0.35 g, yield 51%). IR (KBr) ꢀ: 2967, 2963, 1686, 1634,
2. Experimental
2.1. Instruments and materials
The 1H NMR spectra were recorded at 400 MHz on a Varian
Gemin-400. All absorption spectra were recorded in Pgeneral TU-
1901 UV–Vis spectrometer. All fluorescence measurements were
carried out on Hitachi F-4500 spectrophotometer. All pH values
were measured with a Model pHS-3C pH meter (Shanghai, China).
Thin layer chromatography (TLC) was carried out using silica gel
F254, and column chromatography was conducted over silica gel
(200–300 mesh), both of which were purchased from the Qing-
dao Ocean Chemicals (Qingdao, China). Doubly distilled water was
used for all experiments. The metal ions are perchlorate salts of
Ag+, Cu2+, Zn2+, Cd2+, Pb2+, Hg2+, Fe2+, Fe3+, Co2+, Ca2+, Na+, Ni2+and
Mg2+, which were purchased from Sinopharm Chemical Reagent
Beijing Co., compounds 1, 2, 3 were synthesized and characterized
by the method showed below. All of other chemicals used here were
analytical reagents and used as received.
1615, 1514, 1466, 1421, 1376, 1219, 1120, 817, 747, 704 cm−1 1H
;
NMR (400 MHz, CDCl3), ı (ppm): 1.15 (t, 12H, J = 7.0 Hz, NCH2CH3),
3.27–3.32 (q, 8H, J1 = 7.2 Hz, J2 = 8.0 Hz, NCH2CH3), 4.50 (s, 2H,
CH2-Pyridine), 6.06–6.07 (d, 1H, J = 4.0 Hz, ArH), 6.08–6.09 (d, 1H,
J = 4.0 Hz, ArH), 6.27–6.30 (m, 4H, ArH), 6.83–6.86 (m, 1H, ArH),
7.07–7.09 (d, 1H, J = 8.0 Hz, ArH) 7.12–7.14 (m, 1H, ArH), 7.28–7.32
(m, 1H, ArH), 7.45–7.50 (m, 2H, ArH), 7.98–8.01 (m, 1H, ArH),
8.18–8.20 (m, 1H, ArH); MS (EI) m/z: 533 (M + H+, 100%).
Table 1
The comparison of the properties of current rhodamine-based Cu2+chemosensors.
Sensors
Linear range
Limit of detection
Response time
Working pH
Lit [29]
Lit [31]
Lit [32]
Lit [34]
Lit [35]
2.0 × 10−8–2 × 10−6
M
2.0 × 10−8
2.5 × 10−8
7.8 × 10−7
3.0 × 10−7
3.0 × 10−7
M
M
M
M
M
2 min
7.0
7.0
7.0
6.8
7.1
2.5 × 10−8–3.3 × 10−6
2.0 × 10−7–4.0 × 10−6
6.0 × 10−7–1.0 × 10−4
8.0 × 10−7–1.0 × 10−5
M
M
M
M
–
–
–
1 min

16
Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
N
O
N
H
N
H
H2N
N
N
O
N
N
1
MeOH
H2N
O
N
O
N
Cl
N
N
O
N
MeOH
N
O
N
Cl
H
N
2
H2N
O
MeOH
NH
N
N
O
N
3
Scheme 2. Synthesis and structures of the rhodamine derived compounds: chemosensor 1 and its analogs 2 and 3.
2.4. Synthesis of 3
3. Results and discussion
To a 100 mL flask, rhodamine B acid chloride (0.6 g, 1.3 mmol)
was dissolved in 30 mL methanol. Excess of phenyl hydrazine
(0.36 g, 3.4 mmol) was then added dropwise with vigorous stirring
at room temperature. Then the mixture was heated to reflux for
48 h. The solution changed from dark purple to light orange. Then
the mixture was cooled and solvent was removed under reduced
pressure. 1 M HCl (50 mL) was added to dissolve the residue, then
1 M NaOH (70 mL) was added slowly with stirring until the pH of the
solution reached pH = 9–10. The resulting precipitate was filtered
and washed 3 times with water, and the crude product was puri-
fied by column chromatography (eluant: ethyl acetate/petroleum
ether; v/v = 4/1) to give compound 3 0.31 g as a white solid (yield:
45%). IR (KBr) ꢀ 3434, 3261, 2968, 2926, 1704, 1613, 1515, 1355,
3.1. UV–vis absorption and fluorescence studies of 1 to metal ions
The perchlorate alts of Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Li+, Na+, K+,
Cs+, Fe3+, Fe2+, Cd2+, Cr2+, Hg2+, Mn2+, Ni2+, Pb2+, Sn2+, Zn2+, Ag+ and
Co2+ ions were used to evaluate the metal ion binding properties
of compounds 1, 2, 3 in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1
pH = 7.0) solution. The fluorescence spectra were obtained by exci-
tation of the rhodamine fluorophore at 540 nm. As we expected,
compound 1 (5 ␮M) in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1,
pH = 7.0) solution was colorless and weakly fluorescent (ф= 0.012)
due to the existence of non-conjugated spirolactam structural form
of 1 (Fig. 1a and b). After addition of the perchlorate salts of the
metal ions (50 ␮M) to the solution of 1, a 17-fold significant fluo-
rescence (Fig. 1a) and absorbance (Fig. 1b) enhancement (ф= 0.22)
could be observed with addition of 10 equiv of Cu2+ (Fig. 1), and
a slightly fluorescence enhancement (2-fold) was observed after
addition of Fe3+, and other metal cations did not induce such spec-
troscopic changes. The fluorescent “turn-on” of 1 in the presence
of Cu2+, accompanied an obvious purple color change could be
observed by naked-eyes in aqueous solution, while other metal ions
did not show such color change, indicated the high selectivity of 1
to Cu2+ among the metal cations (Fig. 2).
In order to investigate the dependence of the absorption and
fluorescence intensities to the concentration of Cu2+, we carried
out absorption and fluorescence titration experiments by the addi-
tion of increasing amount of Cu2+ into the aqueous solution of 1. As
shown in Fig. 3a, the fluorescence intensity of 1 (5 ␮M) at 578 nm
was found increased gradually with the increasing of the concentra-
tion of Cu2+ from 0.5 × 10−6 M to 3.0 × 10−6 M and then reached a
platform, indicated a complete complex formation of 1 and Cu2+, as
shown in Fig. 3b, the absorption titration is in accordance with the
result of fluorescence titration. More importantly, a linear relation-
ship between the fluorescence intensity of 1 and Cu2+ concentration
1224, 1118, 816, 789, 692 cm−1 1H NMR (400 MHz, CDCl3), ı
;
(ppm): 1.13–1.17 (t, 12H, J = 8.0 Hz, NCH2CH3), 3.31–3.37 (q, 8H,
J1 = 6.8 Hz, J2 = 7.2 Hz, NCH2CH3), 6.26–6.27 (d, 1H, J = 2.4 Hz, ArH),
6.29–6.30 (d, 1H, J = 2.4 Hz, ArH), 6.50–6.51 (d, 2H, J = 2.4 Hz, ArH),
6.75–6.77 (d, 2H, J = 2.4 Hz, ArH), 7.02–7.03 (m, 1H, ArH), 7.14–7.18
(m, 3H, ArH), 7.25–7.27 (m, 2H, ArH), 7.49–7.50 (m, 2H, ArH),
8.36–8.37 (m, 1H, ArH).
2.5. Absorption and fluorescence spectroscopy measurements
Rhodamine derivatives 1, 2 and 3 was dissolved in ethanol to
prepare the stock solution with a concentration of 10 mM, then
diluted with 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) buffer solu-
tion for spectroscopy measurement. The perchlorate salts of Be2+
Mg2+, Ca2+, Sr2+, Ba2+, Li+, Na+, K+, Cs+, Fe3+, Fe2+, Cd2+, Cr2+, Hg2+
,
,
Mn2+, Ni2+, Pb2+, Sn2+, Zn2+, Ag+ and Co2+ were dissolved in distilled
water to prepare the stock solution with a concentration of 10 mM.
The spectra were recorded in Pgeneral TU-1901 UV–Vis and Hitachi
F-4500 spectrophotometer respectively.

Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
17
0.75
a
b
120
100
80
60
40
20
0
2+
Cu
2+
Cu
0.50
2+ 2+ 2+ 2+ 2+ 2+
Ba ,Be ,Mg ,Ca ,Sr ,Cd
2+ 2+ 2+ 2+ 2+ 2+
Ba ,Be ,Mg ,Ca ,Sr ,Cd
2+ 2+ 2+ 2+ 2+
2+
Co ,Fe ,Ni ,Cr ,Mn ,Pb
2+ 2+ 2+ 2+ 2+
2+
Co ,Fe ,Ni ,Cr ,Mn ,Pb
2+ 2+ 2+
+ +
+
3+
Fe
Ag ,Hg ,Sn ,Zn ,Cs ,K
0.25
0.00
2+ 2+ 2+
+
+
+
Ag ,Hg ,Sn ,Zn
Li ,Na ,1
+
+
+
+
Cs ,K ,Li ,Na ,1only
Fe
550
600
650
700
500
600
Wavelength(nm)
Wavelength (nm)
Fig. 1. Fluorescence spectra (a) and absorption (b) of 1 (5 ␮M) in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) solution in the presence of various metal ions (50 ␮M)
Other ions: Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Li+, Na+, K+, Cs+, Fe3+, Fe2+, Cd2+, Cr2+, Hg2+, Mn2+, Ni2+, Pb2+, Sn2+, Zn2+, Ag+ and Co2+
.
was found with the Cu2+ concentration in the range from 0.5 × 10−6
to 3.0 × 10−6 M (Fig. 4), with a detection limit [36] of 2.11 × 10−8 M
(based on S/N = 3). The results indicate that 1 could be employed
to detect micromolar lever concentration of Cu2+ in aqueous solu-
tion, additionally, the linear relationship between 1 and Cu2+ (from
0.5 × 10−6 to 3.0 × 10−6 M) benefits for the establishment of stan-
dard working curves in practical Cu2+detection.
Fig. 2. Photographs of 10 ␮M 1 after addition of 50 ␮M metal ions in buffered 10 mM
Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) solution, from left to right: Cu2+, Fe3+, Fe2+, Pb2+
Hg+, Cr2+ and Ni2+
,
3.2. Binding constant and stoichiometry
.
In order to investigate the binding capability of 1 to Cu2+, Job’s
plot (1 and Cu2+ with a total concentration of 3.0 ␮M) was utilized
to determine the binding stoichiometry of the 1-Cu2+ complex. It
revealed that 1 and Cu2+ formed a complex in 1:1 stoichiometry (as
60
50
40
30
20
10
a
0.5
60
50
40
30
20
10
0
b
0.4
0.3
0.4
0.2
0.1
0.3
0
1 2 3 4 5 6 7 8
0
1
2
3
4
5
6
7
8
Ccu /10
M
0.2
0.1
0.0
Ccu /10 M
550
600
650
700
500
550
600
650
700
Wavelength(nm)
wavelength(nm)
Fig. 3. Fluorescence spectra (a) and absorption (b) of 1 (5 ␮M) in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) solution in the presence of various amount of Cu2+. Inset:
absorbance at 559 nm (a) and fluorescence intensity at 578 nm (b) in increasing of Cu2+ concentration.
10
10
9
F=2.0589C+3.0039
R2=0.9960
3.0
8
6
4
2
0
8
7
6
5
4
3
0.5 μM
Cu
0.0 0.5 1.0 1.5 2.0 2.5 3.0
550
600
650
700
-6
Ccu2+
/10 M
Wavelength(nm)
Fig. 4. Linear relationships between 1 (1 ␮M) and Cu2+ concentration (from 0 to 3.0 ␮M) by a plot of fluorescence intensity as a function of Cu2+ concentration.

18
Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
0.20
0.16
0.12
0.08
0.04
0.4
0.3
0.2
0.1
50 μM Cu2+
5 μM Cu2+
1 μM Cu2+
0.0
0.0
0.2
0.4
0.6
0.8
1.0
2
4
6
8
10
Cu2+/Cu2++ 1
time (min)
Fig. 5. Job’s plot of 1 and Cu2+ in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0),
(total concentration of 1 and Cu2+ was kept at 3.0 ␮M, absorbance was recorded at
558 nm).
Fig. 7. Absorbance at 558 nm of
1 (5 ␮M) in buffered 10 mM Tris–HCl/EtOH
(v/v = 1/1, pH = 7.0) with addition of different concentration of Cu2+(50 ␮M, 5 ␮M
and1 ␮M).
shown in Fig. 5). On the base of the 1:1 stoichiometry, we calculated
the value of the stability constant for the complex by nonlinear
curve fitness [37] as following expression. Here Y and Y0 are the
value from 7.0 to 4.0, due to the dissociation of coordinated Cu2+ by
the compatible protonation on pyridine and hydrazine moiety; the
highest absorbance response was observed after addition of Cu2+
around pH 7.0, then decreased under higher pH from 8.0 to 10.0,
due to the formation of Cu (OH) 2 under higher pH value and result
in the decrease of the absorbance.
absorbance of the solution of 1 in the presence and absence of Cu2+
,
CM and CL are the concentrations of Cu2+ and 1; Ks is the stability
constant. According to titration data, the stability constant between
1 and Cu2+ was determined as Ks = 3.84 × 105 (r2 = 0.9958).
ꢀ
Ylim − Y0
CM
CL
1
KSCL
ꢄ
3.4. Response time of 1 to Cu2+
Y = Y0
+
1 +
+
−
2
ꢅ
ꢁ
It is known that a short response time is preferred for a high
efficient chemosensor [30]. Herein, the time dependence of absorp-
tion change for 1 to Cu2+ was investigated by recording the change
of absorbance at 558 nm against times under different concentra-
tions of Cu2+. As shown in Fig. 7, the results revealed that the
response time of 1 to Cu2+ increased with the increasing of Cu2+
concentration. The absorbance at 558 nm of 1 (5 ␮M) increased
rapidly and almost reached a platform within 10 min after addi-
tion of 50 ␮M of Cu2+ (line a), while slow response was observed at
lower Cu2+concentration less than 5 ␮M (lines b and c). Moreover,
the short response time of 1 to Cu2+ (>50 ␮M) demonstrates the
possible application of 1 in real-time Cu2+ detection.
1/2
ꢂ
ꢃ
2
CM
1
CM
1 +
+
− 4
CL
KSCL
CL
3.3. Influence of pH value on Cu2+ detection
In many cases, the solution pH strongly affects the spectroscopic
properties or responses of chemosensors [38]. Therefore, the influ-
ence of pH on the response of 1 to Cu2+ was also investigated in
this work. As depicted in Fig. 6, no significant absorbance change at
558 nm of 1 (10 ␮M) could be observed (bottom line) in the pH rang-
ing from 4.0 to 10.0, suggesting that the spirolactam form was stable
within this pH range. However, in the presence of Cu2+ (100 ␮M),
an obvious absorbance change at 558 nm of 1 was observed under
different pH values range from 4.0 to 10.0 (Fig. 6, upper line). The
absorbance at 558 nm decreased drastically with the decrease of pH
3.5. Selectivity of 1 to Cu2+ in the presence of other coexisting
metal cations
Selectivity is a very important parameter for evaluating the per-
formance of a chemosensor. The interference of coexist metal ions
to Cu2+ detection was investigated by UV–vis absorption spectra.
As shown in Table 2, no significant absorbance changes of 1/Cu2+
solution could be observed in the presence of the coexist metal ions
0.6
1+100μ M Cu2+
0.5
Table 2
0.4
0.3
0.2
Absorbance at 558 nm of 1 (5 ␮M) in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1,
pH = 7.0) at the presence of 50 ␮M (10 equiv) Cu2+ and 150 ␮M other metal ions.
Cu2+ + metal ions
Absorbance (at 558 nm)
Cu2+ + Ca2+
Cu2+ + Na+
Cu2+ + Pb2+
Cu2+ + Fe2+
Cu2+ + Cd2+
Cu2+ + Zn2+
Cu2+ + Hg2+
Cu2+ + Mg2+
Cu2+ + Ag+
Cu2+ + Fe3+
Cu2+ + Ni2+
Cu2+
0.440
0.441
0.438
0.440
0.435
0.438
0.441
0.445
0.430
0.450
0.440
0.442
0.1
1
0.0
4
5
6
7
8
9
10
pH value
Fig. 6. Absorbance of 1 (10 ␮M) in Tris–HCl (10 mM) aqueous buffers of different pH
in the absence (bottom line, at 558 nm) and presence (top line, at 558 nm) of 100 ␮M
Cu2+. The change of absorbance for 1 alone is quite mild in pH 5.0–10.0 range.

Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
19
0.4
0.3
0.2
0.1
0.0
b
d
40
30
20
10
0
b
c
a,c
a
400 450 500 550 600 650 700
Wavelength(nm)
550
600
650
700
Wavelength(nm)
Fig. 8. (a) Fluorescence spectra in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) solutions. Line a: 5 ␮M 1. Line b: 5 ␮M 1 + 5 ␮M Cu2+. Line c: 5 ␮M 1 + 5 ␮M Cu2+ + 1 mM
EDTA, line d: 5 ␮M 1 + 5 ␮M Cu2+ + 1 mM EDTA + 1.1 mM Cu2+ (Excitation at 540 nm). (b) Absorption spectra in buffered 10 mM Tris–HCl/EtOH (v/v = 1/1, pH = 7.0) solutions.
Line a: 5 ␮M 1. Line b: 5 ␮M 1 + 5 ␮M Cu2+. Line c: 5 ␮M 1 + 5 ␮M Cu2+ + 1 mM EDTA.
(150 ␮M, 3 times to Cu2+), indicating that 1 exhibits high selectiv-
60
ity to Cu2+ over other metal ions. These results showed that 1 has
remarkable sensing selectivity to Cu2+, which made its suitable of
1+Cu
50
practical Cu2+ detection.
40
3.6. Sensing mechanism
30
For further understanding of the interaction between 1 and Cu2+
,
we studied the chemical reversibility of the binding of 1 to Cu2+ in
buffered solution. As shown in Fig. 8a, the enhancement of the flu-
orescence spectra at 558 nm of 1 induced by Cu2+ could be almost
completely quenched with the addition of excess EDTA, due to the
strong coordination capability of chelating agent EDTA to Cu2+ (line
c). Moreover, after the addition of excess Cu2+ to the above mixed
solution, the fluorescence intensity at 558 nm could recover to 80%
of the original ones. These results indicated that the binding of 1
to Cu2+ is a chemically reversible coordination rather than a metal
cation-catalyzed reaction, and 1 was not cleaved in the process of
Cu2+ binding [29]. On the other hand, UV–vis spectra also illus-
trated the coordination is chemically reversible (Fig. 8b), as seen by
naked-eyes, the purple color of the mixed solution of 1/Cu2+ disap-
peared instantly upon the addition of excess EDTA, similar to many
rhodamine spirolactam-based fluorescent chemosensors reported
formerly [35], the fluorescence enhancement of 1 upon addition of
Cu2+ could be rationalized by the metal ion induced spirolactam
ring-opening mechanism.
20
10
2+Cu
3+Cu
3
2
1
0
550
600
650
700
Wavelength (nm)
Fig. 9. Fluorescence spectra of 1, 2, 3 (2.5 ␮M) in buffered 10 mM Tris–HCl/EtOH
(v/v = 1/1 pH = 7.0), in the absence and presence of 25 ␮M Cu2+
.
relationship of the neighbor groups/atoms of nitrogen atom fur-
ther demonstrate that nitrogen atoms of pyridine 2-position and
hydrazo-play very important roles for the selectivity/sensitivity of
rhodamine-based chemosensors.
4. Fast detection of Cu2+ with the test strips of 1
To further illustrate the structure-recognition activity relation-
ship of the chemosensor 1, two analog compounds 2 and 3 were
designed and synthesized. As shown in Scheme 2, we noticed that
1 bearing a 2-hydrazinopyridine moiety, in which a nitrogen atom
attached to the 2-position of pyridine, 2 have a methylene-pyridine
moiety, lack of a nitrogen atom attached to 2-position of pyri-
dine, while 3 with a phenyl attached to hydrazine moiety instead
of pyridine. The structure differences of 1, 2 and 3 might affect
their binding capability to Cu2+. The comparison of the fluores-
cence intensity of 1, 2 and 3 with addition of Cu2+ under the same
concentration was shown in Fig. 9, a 17-fold florescence enhance-
ment was observed in the solution of 1, while only 3-fold and
4-fold fluorescence enhancement was observed in 2 and 3 solu-
tions respectively. The results indicated that 1 have more effectively
binding capability to Cu2+ than its analogs 2 and 3. Due to the
synergetic effect of the nitrogen atoms on pyridine ring and the
hydrazo-attached to pyridine 2-position improve the Cu2+ coor-
dination capability of 1, while 2 and 3 did not have both of the
nitrogen atoms, that resulted in much less coordination capabil-
ity to Cu2+. Associate to Yuꢀ s reported derivative [31], which a
more methane–carbon atom was linked between the pyridine 2-
position and the hydrazo-rhodamine than that of 1 and also showed
an effectively binding capability to Cu2+, these structure activity
For the requirement of practical application, an easily prepared
and portable detection kit/strips should be taken into consider-
ation, therefore, test strips were prepared by immersing filter
papers into a EtOH/water (v/v = 1/1) solution of 1 (0.1 M) and
then drying in air. The test strips made from 1 were directly uti-
Fig. 10. Photographs of the test strips made from 1 for the detection of Cu2+
with different concentrations in EtOH/water (v/v = 1/1) solution. Left to right: 0,
1.0 × 10−4 M, 5.0 × 10−4 M, 5.0 × 10−3 M.

20
Y. Yang et al. / Spectrochimica Acta Part A 81 (2011) 14–20
lized in the detection of Cu2+ in EtOH/water (v/v = 1/1) solution
under different concentration. The obvious color change of 1 solu-
tion was observed only treated with Cu2+ solution, while other
metal ions solutions do not induce such changes [36]. Moreover,
the test strips were utilized for the sensing of different concen-
tration of Cu2+, as depicted in Fig. 10, the purple-red color of
the test strips intensified from 0 to 1.0 × 10−4 M, and the col-
orimetric changes exhibited that the test strips made from 1
naked-eyes could be employed as portable tools for fast Cu2+
detection.
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In summary, we synthesized and characterized a rhodamine-
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other metal cations. The remarkable fluorescence and absorp-
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detected by spectroscopy, and the obvious color change also
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.05.016.