A new pyrene-based Schiff-base: A selective colorimetric and fluorescent chemosensor for detection of Cu(II) and Fe(III)

Article abstract of DOI:10.1016/j.saa.2014.03.110

A new receptor 1 was prepared, for the detection of Cu²⁺ and Fe³⁺ in solutions as a colorimetric and fluorescent sensor, respectively. Receptor 1 shows highly selective and sensitive recognition toward Cu²⁺ and Fe³⁺ by naked eye UV-Vis and fluorescent color changes in aqueous solution (DMSO/H₂O = 8/2, v/v), respectively. The sensitivity toward Cu²⁺ or Fe³⁺ was not interfered with by the presence of other metal ions such as Mg²⁺, Cd²⁺, Ag⁺, Zn²⁺, Ni²⁺, Co²⁺, Mn ²⁺, Cr³⁺, Ca²⁺, Na⁺, Pb ²⁺, K⁺, Fe²⁺, Li⁺ and Hg ²⁺ ions. Receptor 1 can be used for semi-quantitative recognition of Cu²⁺ ions at ppm level. The fluorescence microscopy experiments showed that the receptor is efficient for detection of Fe³⁺ in vitro, developing a good image of the biological organelles.

Full text of DOI:10.1016/j.saa.2014.03.110

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A new pyrene-based Schiff-base: A selective colorimetric and
fluorescent chemosensor for detection of Cu(II) and Fe(III)
Yeshwant Ramchandra Bhorge a, Haw-Tyng Tsai a, Keh-Feng Huang a, Albert J. Pape a,
Sudhakar Narasimha Janaki b, Yao-Pin Yen a,
⇑
a Department of Applied Chemistry, Providence University, 200, Sec. 7, Taiwan Boulevard, Shalu Dist, Taichung City 43301, Taiwan
b Graduate Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan
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
ꢀ
New Schiff base sensor is selectively
detects Cu2+ ions by naked eye color
change.
ꢀ
ꢀ
Fe3+ ions are also selectively detected
by monitoring fluorescence emission.
The sensing mechanisms observed
are binding and hydrolysis,
respectively.
ꢀ
ꢀ
The binding and hydrolysis products
were confirmed by 1H NMR, MS.
The new sensor has potential future
use in biological cell imaging studies.
a r t i c l e i n f o
a b s t r a c t
A new receptor 1 was prepared, for the detection of Cu2+ and Fe3+ in solutions as a colorimetric and
fluorescent sensor, respectively. Receptor 1 shows highly selective and sensitive recognition toward
Cu2+ and Fe3+ by naked eye UV–Vis and fluorescent color changes in aqueous solution (DMSO/H2O = 8/
2, v/v), respectively. The sensitivity toward Cu2+ or Fe3+ was not interfered with by the presence of other
metal ions such as Mg2+, Cd2+, Ag+, Zn2+, Ni2+, Co2+, Mn2+, Cr3+, Ca2+, Na+, Pb2+, K+, Fe2+, Li+ and Hg2+ ions.
Receptor 1 can be used for semi-quantitative recognition of Cu2+ ions at ppm level. The fluorescence
microscopy experiments showed that the receptor is efficient for detection of Fe3+ in vitro, developing
a good image of the biological organelles.
Article history:
Received 7 January 2014
Received in revised form 20 March 2014
Accepted 29 March 2014
Available online 13 April 2014
Keywords:
Chemosensor
Cu2+ and Fe3+ ions
Pyrene derivative
Colorimetric
Ó 2014 Elsevier B.V. All rights reserved.
Fluorescence
Cell imaging
Introduction
sense more than one cation even in low concentrations [1–5].
Meanwhile, copper and iron cations are very essential ions because
Chemosensors are a well-known kind of receptor that transduce
a chemical signal into action potential through various elements.
The development of chemosensors for sensing Cu2+ and Fe3+ re-
mains an important area in the field of supramolecular chemistry.
Currently, there is an active effort to develop systems, which can
they play important roles in biology, chemistry, and environment.
Iron is an important trace element and the most abundant in the
cells of all organisms [6,7]. The deficiency of Fe3+ causes anemia,
hemochromatosis, liver damage, diabetes, Parkinson’s disease
and cancer [8]. On the other hand, copper is the third most
abundant in the human body and plays an important role in many
physiological systems in organisms [9,10]. If the blood concentra-
⇑
tion of copper falls outside the normal range of 15.7–23.3
[11], it can cause renal problems and Alzheimer’s [12] or
lM
Corresponding author. Tel.: +886 4 2632 8001x15218; fax: +886 4 2632 7554.
E-mail address: ypyen@pu.edu.tw (Y.-P. Yen).
http://dx.doi.org/10.1016/j.saa.2014.03.110
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

8
Y.R. Bhorge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
Parkinson’s [13] diseases. Therefore, the detection of trace amounts
of Cu2+ [14,15] and Fe3+ [16,17] ions is critical. Up to date, many
Schiff-base based chemosensors have been reported for metal ions
including Hg2+ [18], Zn2+ [19], Ca2+ [20] and Pb2+ [21]. However, in
many reports most of them showed fluorescence quenching
response due to the paramagnetic nature of Fe3+ [22,23], which
poses a challenge to develop selective and sensitive fluorescence
turn on Fe3+ receptors. Few dual sensors have been reported for
Cu2+ and Fe3+ [24–26]. To the best of our knowledge, no such
chemosensor for Fe3+ and Cu2+ has been reported.
Cell culture
Human cervical adenocarcinoma cell line HeLa (ATCCÒ CCL-2™)
was used for live cell imaging. The cells were maintained at 37 °C
as a monolayer in Eagle’s Minimum Essential Medium (MEM)
(HyClone) supplemented with 10% fetal bovine serum (HyClone),
100 U/mL of penicillin and 100 lg/mL of streptomycin (HyClone)
in a humidified 5% CO2 incubator. The HeLa cells were seeded on
glass slides and allowed to adhere for 36 h.
Our research work involves the design and synthesis of new
receptors for selective sensing of various metal ions and anions
[27,28]. Recently we reported a new coumarin based Schiff base
chemosensor [29] for Mg2+ and Fe3+ and chemodosimeter [30]
for HSOÀ4 based on hydrolysis of Schiff base. We have now
synthesized a new chemosensor based on hydrolysis of Schiff base
with the help of 1-aminopyrene. This behaves as a dual sensor
upon chemical inputs of Cu2+ and Fe3+ cations over other cations.
Fluorescence imaging
Receptor 1 or Fe(ClO4)3 were dissolved into a 1.0 Â 10À2 M stock
in sterile DMSO and 1.0 Â 10À2 M HEPES buffer mixed at a ratio of
80:20. To examine the fluorescence activity of receptor 1 in live
cells, the HeLa cells were seeded on glass slides and allowed to ad-
here for 36 h. The cells were treated with MEM containing
2 Â 10À5 M Fe(ClO4)3 for 30 min and then washed with PBS three
times. Following washing the cells were loaded with 2 Â 10À5
M
chemosensor 1 in MEM for another 30 min and washed again with
PBS three times to remove the remaining sensor. The fluorescent
images of HeLa cells were captured at wavelength excitation
405 nm by using vertical fluorescence microscope (Olympus
CKX41).
Experimental section
Reagents
In titration experiments, all the cations in the form of perchlo-
rate salts were purchased from Sigma–Aldrich, USA and stored in a
vacuum desiccator. All chemicals were of analytical grade and used
as received with the exception of DMSO, which was distilled over
calcium hydride.
3+
The pH dependence of Cu2+ and Fe response by receptor 1 was
studied by UV–Vis and fluorescence spectroscopy, respectively
The absorption of receptor 1 (5.0 Â 10À5 M) on addition of Cu2+
(4.0 equiv.) at 410 nm was measured and plotted as a function of
pH. The intensity of fluorescence emission of 1 (5.0 Â 10À7 M) at
440 nm on addition of Fe3+ (130.0 equiv.) was similarly plotted.
Measurements were conducted in DMSO/water solution (v/v, 8:2,
0.002 M buffer). The buffers were prepared in water: pH 1–2,
KCl/HCl; pH 3–4, CH3COOH/KOH; pH 5–7, HEPES/HCl; pH 8–9,
Tris/KOH; pH 10–11, NaHCO3/KOH; pH 12, NaCl/KOH.
Instrumentation
Melting points were determined on a Fargo MP-2D melting
point apparatus in open capillaries and are uncorrected. 1H
and 13C NMR were recorded at 400 and 100 MHz on a Bruker spec-
trometer using trimethylsilane (TMS) as an internal standard. The
EI Mass spectra were carried out on a JEOL JMS-SX/SX 102A
Tandem Mass Spectrometer. UV–Vis spectra were performed in
1 cm path length quartz cell using a Cary 300 UV–Vis spectropho-
tometer. Fluorescence spectra were measured with a Perkin Elmer
LS-50B.
Synthesis
Synthesis of (E)-1-((pyren-1-ylimino)methyl)naphthalen-2-ol (1)
To a stirred solution of 1-aminopyrene (4) (0.20 g, 0.92 mmol)
in ethanol (10 ml), 2-hydroxy-1-naphthaldehyde (5) (0.16 g,
0.95 mmol) was added at room temperature. The reaction mixture
was stirred for 12 h. After that, the solid residue was filtered off.
The resulting precipitate was collected and the crude product
was further washed with ethanol several times to afford the pure
product 1. Yield: 0.17 g (51%). mp: 229–230 °C. 1H NMR
(DMSO-d6, 400 MHz) d: 7.20 (d, 1H, J = 9.2 Hz), 7.43 (t, 1H,
J = 7.2 Hz), 7.63 (t, 1H, J = 7.2 Hz), 7.88 (d, 1H, J = 7.6 Hz), 8.05 (d,
1H, J = 9.2 Hz), 8.13 (t, 1H, J = 7.6 Hz), 8.20 (d, 1H, J = 9.2 Hz), 8.25
(d, 1H, J = 8.8 Hz), 8.32-8.37 (m, 3H), 8.45 (d, 1H, J = 8.4), 8.51 (d,
1H, J = 9.2), 8.57 (d, 1H, J = 8.0), 8.68 (d, 1H, J = 8.4), 10.02 (s, 1H),
16.58 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) d: 110.6, 117.6,
121.6, 121.9, 122.3, 124.1, 124.7, 124.9, 125.4, 126.3, 126.7,
127.1, 127.7, 127.9, 128.0, 128.3, 129.1, 129.4, 130.0, 130.3,
131.5, 132.0, 133.9, 137.7, 139.6, 158.6, 169.6; HRMS (EI)
m/z = 371.1308 [M+], calcd for C27H17NO = 371.1310.
UV–Vis titration experiments
The UV–Vis titrations were carried out at 25 °C and concentra-
tion of 5.0 Â 10À5 M with DMSO/H2O (v/v = 8/2, buffered with
HEPES, pH = 7.4). Deionized water and a spectroscopic grade of
DMSO were used as the solvents for the titration experiments
and the cations were diluted to 4.0 Â 10À3 M with DMSO. The
absorbance was measured from 300 to 600 nm, against a blank
in DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4) and
different cation concentrations were added to the 5.0 Â 10À5
M
host solution (4 mL) in portions.
Fluorescence titration experiments
Synthesis of (E)-N-(naphthalen-1-ylmethylene)pyren-1-amine (2)
Receptor 2 was prepared by the same procedure as 1 using
1-aminopyrene (4) and 1-naphthaldehyde (6) in the same molar
ratio; yield: 0.17 g (47%). mp: 156–157 °C. 1H NMR (400 MHz,
DMSO-d6) d: 7.69 (t, 1H, J = 8 Hz), 7.73–7.79 (m, 2H), 8.05–8.25
(m, 7H), 8.30 (d, 2H, J = 7.6 Hz), 8.39 (d, 1H, J = 8 Hz), 8.43 (d, 1H,
J = 7.2 Hz), 8.71 (d, 1H, J = 8.8 Hz), 9.48 (d, 1H, J = 8.4 Hz), 9.53 (s,
1H). 13C NMR (DMSO-d6, 100 MHz) d: 117.0, 123.9, 125.0, 125.4,
The different concentration solutions of cations (4.0 Â 10À3 M)
were introduced in portions to the host (5.0 Â 10À5 M) and the
fluorescence spectra were recorded from 400 to 600 nm at room
temperature each time (excited at 389 nm, with slit widths:
10 nm/5 nm). Fluorescence quantum yields (Uf) were determined
by the comparative method using anthracene
reference standard [31].
(Uf = 0.27) as

Y.R. Bhorge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
9
125.6, 125.7, 126.0, 126.1, 126.6, 126.9, 127.4, 127.5, 128.2, 128.3,
128.8, 129.8, 130.1, 131.8, 131.9, 132.0, 132.3, 133.2, 134.6, 146.4,
162.5; HRMS (EI) m/z = 355.1353 calcd for
[M+],
27H17N = 355.1361.
0.8
0.79
0.78
0.77
0.76
0.75
(b)
1.2
1
(a)
452
349
414
410
480
C
Synthesis of (E)-N-((2-methoxynaphthalen-1-yl)methylene)pyren-1-
amine (3)
0
2
0.8
0.6
0.4
0.2
0
Cu2+ equiv.
Receptor 3 was prepared by the same procedure as 1 using
1-aminopyrene (4) and 2-methoxy-1-naphthaldehyde (7) in the
same molar ratio; yield: 0.18 g (48%). mp: 156–157 °C. 1H NMR
(400 MHz, DMSO-d6): 1H NMR (DMSO-d6, 400 MHz) d: 4.06 (s,
3H), 7.53 (t, 1H, J = 8 Hz), 7.65 (d, 1H, J = 9.2 Hz), 7.75 (t, 1H,
J = 6.8 Hz), 7.94 (d, 1H, J = 8.4 Hz), 8.02 (d, 1H, J = 8.4 Hz), 8.09 (t,
1H, J = 7.6 Hz), 8.15 (d, 1H, J = 8.8 Hz), 8.24 (t, 3H, J = 9.6 Hz), 8.29
(dd, 2H, J = 4.4 Hz, 7.2 Hz), 8.38 (d, 1H, J = 8 Hz), 8.61 (d, 1H,
J = 9.2 Hz), 9.51 (s, 1H), 9.83 (d, 1H, J = 8.8 Hz). 13C NMR (CDCl3,
100 MHz) d: 29.7, 56.7, 112.7, 115.9, 117.3, 123.8, 124.4, 124.8,
125.0, 125.1, 125.3, 125.7, 126.3, 126.5, 127.0, 127.4, 128.4,
128.8, 129.2, 129.3, 131.6, 131.6, 132.1, 134.2, 147.7, 159.4,
0 eq.
2 eq.
550
300
350
400
450
500
600
Wavelength (nm)
160.0;
HRMS
(EI)
m/z = 385.1463
[M+],
calcd
for
Fig. 1. (a) Family of spectra taken in the course of the titration of 1 (5.0 Â 10À5 M)
in DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4) with a standard solution of
Cu2+ at 25 °C. (b) Titration profile (inset) indicates the formation of a 1:1 complex.
C28H19NO = 385.1467.
Results and discussion
Preparation of receptors 1, 2 and 3 is depicted in Scheme 1.
1-Aminopyrene and 2-hydroxynapthaldehyde, 1-naphthaldehyde
and 2-methoxynapthaldehyde were reacted, affording Schiff bases
1, 2, and 3 respectively. All of these compounds were characterized
by 1H NMR, 13C NMR, and HRMS.
(a)1.4
1.2
Host and other ions
1
Fe3+
0.8
0.6
Cu2+
0.4
UV–Vis titration studies
0.2
0
350
400
450
500
550
600
To examine the feasibility of receptor 1 as a chemosensor for
Cu2+, we first investigated its chromogenic character in the absence
and presence of Cu2+ ions. The receptor 1 was stable in a solvent
mixture DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4).
The yellow color solution of 1 is stable within a pH range of 5–9
(Fig. S1b). In the UV–Vis titration of Cu2+ ions, the receptor 1
showed characteristic absorption peaks at 355 and 452 nm respec-
tively. Upon treatment with Cu2+ ions, the absorption peaks
initially at 355 and 452 nm were slowly decreased and the peaks
were blue-shifted to 326 and 410 nm with two isosbestic points
at 349 and 414 nm, respectively (Fig. 1a). The color of the solution
changed from yellow to pale lemon (Fig. 2b). The signaling was
Wavelength (nm)
(b)
2+
Host Mg2+ Cd2+ Ag+ Zn2+ Cu2+ Ni2+ Co2+ Fe3+ Mn2+ Cr3+ Ca2+ Na+ Pb2+ K+ Fe2+ Li+ Hg
Fig. 2. (a) UV–Vis spectra of 1 (5.0 Â 10À5 M) in DMSO/H2O (v/v = 8/2, buffered
with HEPES, pH = 7.4) in the presence 4.0 equiv. of the Cu2+ ion and other cations
including Mg2+, Cd2+, Ag+, Zn2+, Ni2+, Co2+, Fe3+ Mn2+, Cr3+, Ca2+, Na+, Pb2+, K+, Fe2+, Li+
and Hg2+, respectively. (b) Color changes of 1 (5.0 Â 10À5 M) upon addition of 4.0
equiv. cations.
completed within 1 min (Fig. S1a). By plotting the changes in the
absorbance intensity of 1 at 410 nm as a function of Cu2+ concen-
tration, a sigmoidal curve was obtained and is shown in the inset
of Fig. 1b. This indicates a 1:1 stoichiometry between the host 1
and the Cu2+ ions.
O
HO
OH
5
N
Ethanol, rt
1
Competition experiments
O
NH2
An important feature of the chemosensor is its high selectivity
toward the analyte over other competitive species. Variations of
UV–Vis spectral and visual color changes of 1 in DMSO/H2O
(v/v = 8/2, buffered with HEPES, pH = 7.4) solutions caused by mis-
N
6
2
Ethanol, rt
O
cellaneous cations including Mg2+, Cd2+, Ag+, Zn2+, Cu2+, Ni2+, Co2+
,
Fe3+, Mn2+, Cr3+, Ca2+, Na+, Pb2+, K+, Fe2+, Li+ and Hg2+ were recorded
in Fig. 2. Miscellaneous competitive cations caused relatively
minor spectral changes. However, in the presence of miscellaneous
competitive cations, the Cu2+ ions still resulted in a similar UV–Vis
absorption profile (Fig. 3).
4
H3CO
OCH3
7
N
3
Ethanol, rt
The distinct color change of 1 was caused by the Cu2+-induced
formation of 1-Cu2+-DMSO complex as depicted in Scheme 2. To
Scheme 1. Synthetic route of compound 1, 2 and 3.

10
Y.R. Bhorge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
0.8
1
0.8
0.6
0.4
0.2
0
y = 713.16x + 0.7616
0.795
R² = 0.9913
0.79
1+Xn+
1+Xn++Cu2+
0.785
0.78
0.775
0.77
0.765
0.76
Fig. 3. UV absorption response of 1 (5.0 Â 10À5 M) to various cations (4.0 equiv.) in
0.755
DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4). Bars represent the intensity
0
0.00001 0.00002 0.00003 0.00004 0.00005
[Cu2+] [M]
ratio of the absorption at 410 nm and were calculated according to: A =
n+
A
À AHost and A0 = AHost+Cu(II) À AHost
.
Host+X
Fig. 4. Absorbance of 1 versus Cu2+ concentrations [1] = 5.0 Â 10À5 M.
support this supposition, we have carried out 1H NMR titration
experiment for receptor 1 in DMSO-d6 by stepwise addition of
equivalents of Cu2+ ions as perchlorate salts in DMSO/H2O
(v/v = 8/2, buffered with HEPES, pH = 7.4). It was found that when
receptor 1 formed a complex with Cu2+, the proton signal of OH
became broad and slightly shifted downfield with increasing Cu2+
concentration from 0 to 2 equivalents (Fig. S2a). Further, we
proved the formation of 1-Cu2+-DMSO complex by HRMS
(Fig. S2b).
The colorimetric detection limit of 1 for Cu2+ ions was also
tested (Fig. 4). In the solvent system of DMSO/H2O (v/v = 8/2,
buffered with HEPES, pH = 7.4), the detection limit calculated on
the basis of 3
r
/K [32] is (2.17 0.02) Â 10À6 M.
Fluorescence studies
On the other hand, the fluorogenic behavior of 1 was investi-
gated in the absence and presence of Fe3+ ions as shown in Fig. 5.
The receptor 1 was stable in a mixture solvent of DMSO/H2O
(v/v = 8/2, buffered with HEPES, pH = 7.4). The fluorescence blue
color solution of 1 is stable within a pH range of 5–8 (Fig. S3b).
The fluorescence enhancement induced by the addition of Fe3+ ions
was observed and the signaling was complete within 1 min
(Fig. S3a). Upon incremental addition of Fe3+, the initial weak emis-
sion peak at 438 nm was gradually increased and a marked
enhancement in the fluorescence intensity (11-fold) was observed.
The fluorescence color changed from blue to cyan (Fig. 6b). No
obvious spectral changes could be observed when other cations
like Mg2+, Cd2+, Ag+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, Cr3+, Ca2+, Na+,
Pb2+, K+, Fe2+, Li+ and Hg2+ were added (Fig. 6a). Moreover, in the
presence of miscellaneous competitive cations, the Fe3+ ions still
resulted in the similar fluorescence changes (Fig. 7). The increase
of fluorescence intensity resulting from the addition of the Fe3+
ions was not influenced by the subsequent addition of miscella-
neous cations. All these indicate the selectivity of 1 for the Fe3+ ions
over other competitive cations in the aqueous medium is remark-
ably high.
Fig. 5. Fluorescent titration of 1 (5 Â 10À7 M) in DMSO/H2O (v/v = 8/2, buffered
with HEPES, pH = 7.4) with Fe3+ ions.
generate 1-aminopyrene (2) and 2-hydroxynapthaldehyde (3) as
depicted in Scheme 2. The hydrolytic reaction of the Schiff base
promoted by Fe3+ in aqueous media is known to be the key step
towards the sensing [33]. To prove the above process chemodosi-
metric reaction was carried out in between receptor 1 and Fe3+
.
The hydrolysis products, 1-aminopyrene (2) and 2-hydroxynapth-
aldehyde (3) were separated, isolated and confirmed by 1H NMR
spectroscopy, ESI-MS analysis and comparison with authentic
compounds (Fig. S4a–c). The fluorescence quantum yield (Uf) is
0.22 determined after the addition of Fe3+ to the solution of the
receptor 1.
In the solvent system of DMSO/H2O (v/v = 8/2, buffered with
HEPES, pH = 7.4), the detection limit of the fluorescence calculated
on the basis of 3
r
/K [32] is (3.19 0.02) Â 10À6 M (Fig. S5).
Furthermore, in order to investigate how the different substitu-
ents on the phenyl ring of the imine group can influence the Cu2+
and Fe3+ ions sensing properties, the 2-hydroxy-1-naphthaldehyde
(5) was replaced by 2-methoxy-1-naphthaldehyde (7) and
The large fluorescence enhancement of receptor 1 was caused
by the coordination of Fe3+ to the Schiff base first and then
followed by hydrolytic cleavage of the Schiff base linkage to
NH2
Fe3+
Cu2+
O
O
Cu2+
OH
N
N
+
1
OH
O
S
5
4
Scheme 2. The proposed mechanism for sensing of Cu2+ and Fe3+ by 1.

Y.R. Bhorge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
11
1-naphthaldehyde (6). Unfortunately, receptors 2 and 3 both
showed no significant colorimetric and fluorescence spectral
change upon addition of cations, possibly due to lack of accessible
coordinating sites.
(a)
400
350
300
250
200
150
100
50
Fe3+
Applications
Host and other ions
Semi-quantitative determination of Cu2+ in aqueous medium
Given the importance of Cu2+ in biology and industry, it is
essential to develop methods of visual determination in the natural
environment. We prepared various concentrations (10–200 ppm)
of Cu2+ ions in aqueous solution and added to the solution of
chemosensor 1 (5.0 Â 10À5 M) in DMSO/H2O (v/v = 8/2, buffered
with HEPES, pH = 7.4). Receptor 1 showed instant color change in
the presence of various concentrations of Cu2+ ions. This indicates
that receptor 1 can be used for semi-quantitative recognition of
Cu2+ ions at the ppm level as depicted in Fig. 8.
0
405
425
445
465
485
505
Wavelength (nm)
(b)
Host Mg2+ Cd2+ Ag+ Zn2+ Cu2+ Ni2+ Co2+ Fe3+ Mn2+ Cr3+ Ca2+ Na+ Pb2+ K+ Fe2+ Li+ Hg2+
Fig. 6. (a) Fluorescence spectra of 1 (5 Â 10À7 M) with different cations (130 equiv.)
in DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4). (b) Fluorescent changes of
1 (5 Â 10À7 M) upon addition of 130 equiv. of cations in aqueous solution of DMSO/
H2O (v/v = 8/2, buffered with HEPES, pH = 7.4).
Live cell imaging
Receptor 1 was also used for live cell imaging. For the detection
of Fe3+ in living cells, HeLa cells were treated with 2 Â 10À5
M
Fe(ClO4)3 for 30 min and washed with PBS three times. Following
washing the cells were loaded with 2 Â 10À5 M receptor 1 in
MEM for another 30 min and washed again with PBS three times
to remove the remaining receptor. The fluorescent images of HeLa
cells were captured by using vertical fluorescence microscope.
Fig. 9 shows the fluorescence emission of receptor 1 in HeLa
cells after treatment with Fe(ClO4)3. The merger of fluorescence
and phase contrast images shows that the fluorescence signals
are localized in the intracellular region, indicating a subcellular
distribution of Fe3+ and good cell-membrane permeability of
receptor 1.
Fig. 7. Fluorescence intensity response of 1 (5.0 Â 10À7 M) to various cations (130
equiv.) in DMSO/H2O (v/v = 8/2, buffered with HEPES, pH = 7.4). Bars represent the
intensity ratio of the emission at 440 nm and were calculated according to:
n+
I = I
À IHost and I0 = IHost+Fe(III) À IHost
.
Host+X
Fig. 8. Visual determination of 10–200 ppm of Cu2+ in aqueous solutions by using chemosensor 1 (5.0 Â 10À5 M).
Fig. 9. Fluorescence images of HeLa cells treated with 1 and Fe3+. (Left) bright field image; (center) fluorescence image; and (right) merged image.

12
Y.R. Bhorge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 7–12
[9] K.M.K. Swamy, S.K. Ko, S.K. Kwon, H.N. Lee, C. Mao, J.M. Kim, K.H. Lee, J. Kim, I.
Conclusion
Shin, J. Yoon, Chem. Commun. (2008) 5915–5917.
[10] P. Grandini, F. Mancin, P. Tecilla, P. Scrimin, U. Tonellato, Angew. Chem. Int. Ed.
38 (1999) 3061.
In conclusion, pyrene based new Schiff bases 1, 2 and 3 were
[11] W.T. Tak, S.C. Yoon, KSN 20 (2001) 863.
synthesized from 2-hydroxynapthaldehyde, 1-napthaldehyde and
2-methoxynapthaldehyde for recognition of Cu2+ and Fe3+. Schiff
base 1 showed high selectivity toward Cu2+ and Fe3+ over miscella-
neous competitive cations by color and fluorescent changes,
respectively. Fluorescence enhancement at 440 nm was due to
the presence of strong fluorescence of 1-aminopyrene after hydro-
lysis of Schiff base 1 in DMSO/H2O (v/v = 8/2, buffered with HEPES,
pH = 7.4). This receptor is also an effective method for Fe3+ sensing
in live cell imaging. This obvious color and fluorescence change for
Cu2+ and Fe3+ respectively, indicate that this receptor could be em-
ployed as a dual colorimetric-fluorescent probe for monitoring
Cu2+ and Fe3+ ions in biological and environmental systems.
[12] G. Multhaup, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert, C.L. Masters, K.
Beyreuther, Science 271 (1996) 1406.
[13] D. Waggoner, T.B. Bartnikas, J.D. Gitlin, Neurobiol. Dis. 6 (1999) 221.
[14] S.P. Wu, T.H. Wang, S.R. Liu, Tetrahedron 66 (2010) 9655–9658.
[15] E.J. Jun, H.N. Won, J.S. Kim, K.H. Lee, J. Yoona, Tetrahedron Lett. 47 (2006)
4577–4580.
[16] M. She, Z. Yang, B. Yin, J. Zhang, J. Gu, W. Yin, J. Li, G. Zhao, Z. Shi, Dyes Pigm. 92
(2012) 1337–1343.
[17] V.K. Bhardwaj, P. Saluja, G. Hundal, M.S. Hundal, N. Singh, D.O. Jang,
Tetrahedron 69 (2013) 1606–1610.
[18] (a) Z. Dong, X. Tian, Y. Chen, J. Hou, J. Ma, RSC Adv. 3 (2013) 2227–2233;
(b) Z. Dong, X. Tian, Y. Chen, Y. Guo, J. Ma, RSC Adv.
1088.
3 (2013) 1082–
[19] Y. Zhou, H.N. Kim, J. Yoon, Bioorg. Med. Chem. Lett. 20 (2010) 125–128.
[20] J.M. An, Z.Y. Yang, M.H. Yan, T.R. Li, J. Lumin. 139 (2013) 79–83.
[21] Y. Xiang, A. Tong, Y. Lu, J. Am. Chem. Soc. 131 (2009) 15352.
[22] N. Singh, N. Kaur, J. Dunn, M. MacKay, J.F. Callan, Tetrahedron Lett. 50 (2009)
953–956.
[23] J. Yao, W. Dou, W. Qin, W. Liu, Inorg. Chem. Commun. 12 (2009) 116–118.
[24] L. Yang, N.W. Zhu, M. Fang, Q. Zhang, C. Li, Spectrochim. Acta Part A Mol.
Biomol. Spectrosc. 109 (2013) 186–192.
[25] B.K. Paul, S. Kar, N. Guchhait, J. Photochem. Photobiol., A 220 (2011) 153–163.
[26] V. Bravo, S. Gil, A.M. Costero, M.N. Kneeteman, U. Llaosa, P.M.E. Mancini, L.E.
Ochando, M. Parra, Tetrahedron 68 (2012) 4882–4887.
[27] (a) Y.K. Tsui, S. Devaraj, Y.P. Yen, Sens. Actuators B: Chem. 161 (2012) 510–
519;
Acknowledgement
We thank the National Science Council of the Republic of China
(Taiwan) for the financial support.
Appendix A. Supplementary material
(b) Z.H. Liu, S. Devaraj, C.R. Yang, Y.P. Yen, Sens. Actuators B: Chem. 174 (2012)
555–562;
(c) W.C. Lin, Y.P. Tseng, C.Y. Lin, Y.P. Yen, Org. Biomol. Chem. 15 (2011) 5547–
5553.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.03.110.
[28] (a) Y.P. Yen, Y.S. Lin, G.M. Tu, C.Y. Lin, Y.T. Chang, New J. Chem. 33 (2009) 860–
867;
References
(b) Y.P. Yen, Y.P. Tseng, C.H. Lin, Sens. Lett. 6 (2008) 826–830;
(c) Y.P. Yen, Org. Biomol. Chem. 22 (2007) 3592–3598;
(d) Y.P. Yen, K.W. Ho, Tetrahedron Lett. 47 (2006) 1193–1196.
[29] S. Devaraj, Y.K. Tsui, C.Y. Chiang, Y.P. Yen, Spectrochim. Acta Part A Mol.
Biomol. Spectrosc. 96 (2012) 594–599.
[30] C.Y. Lin, K.F. Huang, Y.P. Yen, Spectrochim. Acta Part A Mol. Biomol. Spectrosc.
115 (2013) 552–558.
[31] A.M. Brouwer, Pure Appl. Chem. 83 (2011) 2213–2228.
[32] J. Liu, Y. Lu, Angew. Chem. Int. Ed. 46 (2007) 7587–7590.
[33] V. Kumar, A. Kumar, U. Diwan, K.K. Upadhyay, Chem. Commun. 48 (2012)
9540–9542.
[1] T.B. Wei, Z. Zhang, B.B. Shi, P. Chen, Q. Lin, J. Liu, Y.M. Zhang, Dyes Pigm. 97
(2013) 297.
[2] A. Sikdar, S.S. Panja, P. Biswas, S. Roy, J. Fluoresc. 22 (2012) 443–450.
[3] V. Bravo, S. Gil, A.M. Costero, M.N. Kneeteman, U. Llaosa, P.M.E. Mancini, L.E.
Ochando, M. Parra, Tetrahedron 68 (2012) 4882.
[4] B.K. Paul, S. Kar, Guchhait, J. Photochem. Photobiol., A 220 (2011) 153–163.
[5] Y.K. Jang, U.C. Nama, H.L. Kwon, I.H. Hwang, C. Kim, Dyes Pigm. 99 (2013) 6.
[6] Y. Xing, A. Ton, Org. Lett. 8 (2006) 1549.
[7] G.E. Tumambac, C.M. Rosencrance, C. Wolf, Tetrahedron 60 (2004) 11293.
[8] S. Narayanaswamy, T. Govindaraju, Sens. Actuators B 161 (2012) 304.