A pyrene-based highly selective turn-on fluorescent sensor for copper(II) ion and its application in live cell imaging

Article abstract of DOI:10.1007/s10895-011-0955-7

A new pyrene derivative (chemosensor 1) containing a picolinohydrazide moiety exhibits high selectivity for Cu²⁺ ion detection in mixed aqueous media (CH3OH:H2O= 7:3). Significant fluorescence enhancement was observed with chemosensor 1 in the presence of Cu²⁺. However, the metal ions Ag+, Ca²⁺, Cd²⁺, Co²⁺, Cu ²⁺, Fe²⁺, Fe³⁺, Hg²⁺, K+, Mg ²⁺, Mn²⁺, Ni²⁺, Pb²⁺, and Zn ²⁺ produced only minor changes in fluorescence for the system. The apparent association constant (Ka) of Cu²⁺ binding in chemosensor 1 was found to be 2.75103 M-1. The maximum fluorescence enhancement caused by Cu²⁺ binding in chemosensor 1 was observed over the pH range 5-8. Moreover, by means of fluorescence microscopy experiments, it is demonstrated that 1 can be used as a fluorescent probe for detecting Cu²⁺ in living cells.

Full text of DOI:10.1007/s10895-011-0955-7

J Fluoresc (2012) 22:253–259
DOI 10.1007/s10895-011-0955-7
ORIGINAL PAPER
A Pyrene-based Highly Selective Turn-on Fluorescent Sensor
for Copper(II) Ion and its Application in Live Cell Imaging
Shu-Pao Wu & Zhen-Ming Huang & Shi-Rong Liu &
Peter Kun Chung
Received: 20 June 2011 /Accepted: 10 August 2011 /Published online: 26 August 2011
#
Springer Science+Business Media, LLC 2011
Abstract A new pyrene derivative (chemosensor 1) contain-
ing a picolinohydrazide moiety exhibits high selectivity for
Cu2+ ion detection in mixed aqueous media (CH3OH:H2O=
7:3). Significant fluorescence enhancement was observed with
chemosensor 1 in the presence of Cu2+. However, the metal
ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+,
Mn2+, Ni2+, Pb2+, and Zn2+ produced only minor changes in
fluorescence for the system. The apparent association constant
(Ka) of Cu2+ binding in chemosensor 1 was found to be
2.75*103 M−1. The maximum fluorescence enhancement
caused by Cu2+ binding in chemosensor 1 was observed over
the pH range 5–8. Moreover, by means of fluorescence
microscopy experiments, it is demonstrated that 1 can be used
as a fluorescent probe for detecting Cu2+ in living cells.
disease [3], and Menkes and Wilson diseases [4, 5]. Due to
its extensive applications, the copper ion is also a significant
metal pollutant. The limit of copper in drinking water set by
the US Environmental Protection Agency (EPA) is 1.3 ppm
(~20 μM). Numerous methods for the detection of copper
ions in a sample have been proposed, including atomic
absorption spectrometry [6], inductively coupled plasma mass
spectroscopy (ICPMS) [7], inductively coupled plasma-
atomic emission spectrometry (ICP-AES) [8], and voltametry
[9]. Most of these methods cannot be used for assays because
they entail the destruction of the sample. Consequently, more
attention is being focused on the development of fluorescent
chemosensors for the detection of Cu2+ ions [10–24].
Developing metal ion fluorescent chemosensors usually
involves combining a metal-binding unit with a fluorophore.
The presence of metal ions is signaled, during interaction with
binding units, by changes in emission intensity or wavelength.
Because Cu2+ is known as a fluorescence quencher, most
fluorescent chemosensors detect Cu2+ through a fluorescence-
quenching process that undergoes a charge or energy transfer
mechanism [20]. However, fluorescent chemosensors using
fluorescence enhancement are more sensitive to metal ions
than are those using fluorescence quenching. This paper
reports on a newly designed pyrene-based fluorescent
enhancement chemosensor for Cu2+ based on photoinduced
electron transfer (PET). Binding Cu2+ to the chemosensor
blocks PET and greatly enhances fluorescence of pyrene.
In this study, a pyrene-based fluorescent chemosensor was
designed for metal ion detection. Two parts make up chemo-
sensor 1: a pyrene moiety as a reporter and a picolinohy-
drazide as a chelator for the metal ion. Chemosensor 1 was
synthesized through the reaction of 1-pyrenecarboxaldehyde
hydrazone and picolinyl chloride (Scheme 1). Chemosensor 1
exhibits weak fluorescence due to fluorescence quenching by
photoinduced electron transfer from the lone pair of electrons
Keywords Cu2+ Fluorescence Chemosensor Pyrene
.
.
.
.
Live cell imaging
Introduction
Copper, after iron and zinc, is the third most abundant
essential transition metal ion in the human body and plays
important roles in various biological processes. Many proteins
contain copper ions as part of a catalytic center. Free copper
ions in a live cell catalyze the formation of reactive oxygen
species (ROS) that can damage lipids, nucleic acids, and
proteins. Research has connected the cellular toxicity of
copper ions with serious diseases including Alzheimer’s
disease [1], Indian childhood cirrhosis (ICC) [2], prion
:
:
:
S.-P. Wu (*) Z.-M. Huang S.-R. Liu P. K. Chung
Department of Applied Chemistry,
National Chiao Tung University,
Hsinchu, Taiwan 300, Republic of China
e-mail: spwu@mail.nctu.edu.tw

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J Fluoresc (2012) 22:253–259
Scheme 1 The synthesis of
chemosensor 1
O
H
N
NH2
N
N
N
O
Cl
O
N
N2H4
THF, r.t.
THF, Et3N
1
on the nitrogen atom to pyrene. Binding metal ions to the
chemosensor blocks PET and greatly enhances fluorescence
of pyrene. The metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+,
Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+ were
tested for metal ion binding selectivity with chemosensor 1,
but Cu2+ was the only ion that caused a blue emission upon
binding with chemosensor 1. The fluorescence microscopy
experiments also demonstrated that 1 can be used as a
fluorescent probe for detecting Cu2+ in living cells.
7.8 Hz), 8.22 (4H, m), 8.12 (1H, t, J=7.8 Hz), 8.04 (1H, t, J=
7.8 Hz), 7.94 (1H, td, J=1.8, 7.8 Hz), 7.50–7.55 (1H, m). 13C
NMR (75 MHz, CDCl3) δ 160.5, 149.6, 148.5, 147.6, 138.1,
133.3, 131.7, 131.0, 129.9, 129.3, 129.0, 127.9, 127.2, 126.7,
126.6, 126.4, 126.2, 126.1, 125.5, 125.2, 125.0, 123.4, 122.6.
IR (KBr) 3462 (NH), 1667 (C=O), 1587 (C=C, C=N) cm−1.
Metal Ion Binding Study by UV-vis and Fluorescence
Spectroscopy
Chemosensor 1 (25 μM) was added with different metal
ions (50 μM). All spectra were measured in 1.0 mL
methaol-water solution (v/v=7/3, 6 mM Hepes buffer, pH
7.0). The light path length of cuvvet was 1.0 cm.
Experimental Section
Materials and Instrumentations
The pH Dependence on Cu2+ Binding in Chemosensor 1
Studied by Fluorescence Spectroscopy
All solvents and reagents were obtained from commercial
sources and used as received without further purification.
UV/Vis spectra were recoreded on an Agilent 8453 UV/Vis
spectrometer. IR data were obtained on Bomem DA8.3
Fourier-Transform Infrared Spectrometer. NMR spectra
were obtained on a Bruker DRX-300 NMR spectrometer.
Fluorescence imagings were obtained on a ZEISS Axio
Scope A1 Fluorescence Microscope.
Chemosensor 1 (25 μM) was added with Cu2+ (100 μM) in
1.0 mL methaol-water solution (v/v=7/3, 6 mM buffer).
The buffers were: pH 1 ~2, KCl/HCl; pH 2.5~4, KH2PO4/
HCl; pH 4.5~6, KH2PO4/NaOH; pH 6.5~10 Hepes.
Determination of the Binding Stochiometry
and the Apparent Association Constants Ka of Cu(II)
Binding in Chemosensor 1
Synthesis of [N′-(pyren-3-yl)methylene]picolinohydrazide
(chemosensor 1)
The binding stochiometry of 1-Cu2+ complexes was
determined by Job plot experiments [25]. The fluorescence
intensity at 455 nm was plotted against molar fraction of 1
under a constant total concentration. The total concentration
of sensor and Cu2+ ion was 25μM. When the emission
intensity reaches a maximum point, the molar fraction
represents the binding stochiometry of 1-Cu2+ complexes.
In Fig. 4, maximum emission intensity was reached when
the molar fraction was 0.5. These results indicate that
chemosensor 1 forms a 1:1 complex with Cu2+. The
apparent association constants (Ka) of 1-Cu2+ complexes
was determined by the Benesi-Hildebrand Eq. 1 [26, 27]:
The reaction mixture containing picolinic acid (135.42 mg,
1.1 mmol) and thionyl chloride (0.146 ml, 2mmol) in 10 mL
of CH3CN was reflux for one hour. The solvent was removed
by rotor vacuum, and THF was added to dissolve the product
(picolinoyl chloride). 1-Pyrenecarboxaldehyde hydrazone
(244 mg, 1 mmol) and triethylamine (0.418 ml, 3 mmol)
were added to the previous solution containing picolinyl
chloride. The reaction mixture was stirred at room tempera-
ture for four hours. Thereafter, the solvent was evaporated
under reduced pressure, and the crude product was purified
by column chromatography (hexane/dichloromethane/ethyl
acetate=2:1:1) to give chemosensor 1 as a pale yellow solid.
Yield: 47%; mp, 252 °C. EI-Mass m/z (%), 349 (16.12%),
227 (100%), 213 (24.68%); HR-MS (EI) calcd for
C23H15O1N3 [M]+ 349.1215; found 349.1208. 1H NMR
(300 MHz, CDCl3): δ 11.25 (1H, s) , 9.37 (1H, s), 8.77 (2H,
d, J=7.8 Hz), 8.65 (1H, d, J=4.8 Hz), 8.38 (1H, d, J=
È
Â
ÃÉ
1=ðF ꢀ F0Þ ¼ 1= Ka ðFmax ꢀ F0Þ Cu2þ þ 1=ðFmax ꢀ F0Þ
ð1Þ
»
»
, where F is the fluorescence intensity at 455 nm at any
given Cu2+ concentration, F0 is the fluorescence intensity

J Fluoresc (2012) 22:253–259
255
at 455 nm in the absence of Cu2+, and Fmax is the maxima
fluorescence intensity at 455 nm in the presence of Cu2+ in
solution. The association constant Ka was evaluated
graphically by plotting 1/(F-F0) against 1/[Cu2+]. Typical
plots (1/(F-F0) vs. 1/[Cu2+] ) are shown in Fig. 5. Data
were linearly fitted according to Eq. 1 and the Ka value
was obtained from the slope and intercept of the line.
Fluorescence Microscope. Cells loaded with 1 were
excited at 350 nm using a lamp (Hg 50 W). Emission
was collected at 460 nm.
Results and Discussion
Spectral Characteristics of Chemosensor 1
Cell Culture
The synthesis of chemosensor 1 consisted of two steps
(scheme 1): the formation of 1-pyrenecarboxaldehyde hydra-
zone and its further reaction with picolinoyl chloride.
Chemosensor 1 is colorless and has an absorption band
centered at 360 nm, which is near the typical absorption band
of pyrene, 335 nm [28]. In addition, chemosensor 1 exhibits
weaker fluorescence (Φ=0.013) than does pyrene (Φ=
0.6~0.9) [29]. This is due to fluorescence quenching by PET
from the lone pair of electrons on the nitrogen atom to pyrene.
The cell line HeLa was provided by the Food Industry
Research and Development Institute (Taiwan). The HeLa
cells were grown in DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% FBS (fetal bovine serum)
at 37 °C and 5% CO2. Cells were plated on 14 mm glass
coverslips and allowed to adhere for 24 h.
Fluorescence Imaging
Experiments to assess Cu2+ uptake were performed in PBS
with 10 μM CuCl2. Treat the cells with 2 μL of 10 mM metal
ions (final concentration: 10 μM) dissolved in sterilized PBS
(pH 7.4 ) and incubate for 0.5–1 h at 37 °C.Wash the treated
cells three times with 2 mL PBS to remove the
remaining metal ions. Add 2 mL culture media to the
cell culture and treat the cell culture with 2 μL of
10 mM chemosensor 1 (final concentration: 10 μM)
dissolved in DMSO. Incubate for 30 min at 37 °C.
Remove culture media and wash the treated cells three
times with 2 mL PBS before observation. Fluorescence
imaging was performed with a ZEISS Axio Scope A1
Cation-sensing Properties
The sensing ability of chemosensor 1 was tested by mixing
it with the metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+,
Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+. To
further evaluate the selectivity of chemosensor 1 toward
various metal ions, the fluorescence spectra of chemosensor
1 were taken in the presence of several transition metal
ions. Cu2+ was the only metal ion that caused a significant
blue emission (Fig. 1). During Cu2+ titration with chemo-
sensor 1, a new emission band centered at 455 nm formed
(Fig. 2). After adding two equivalents of Cu2+, the emission
Fig. 1 Fluorescence emission
(top) and spectra (bottom) of
chemosensor 1 (25 μM) upon
addition of various metal ions
(50 μM) in methanol-water
(v/v=7:3, 6 mM HEPES, pH
7.0) solutions. The excitation
wavelength was 360 nm
600
1 + Cu2+
500
400
300
200
100
0
1 , Ag+, Ca2+, Cd2+, Co2+,
Fe2+, Fe3+, Hg2+, K+, Mg2+,
Mn2+, Ni2+, Pb2+, Zn2+
400
500
600
Wavelength (nm)

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J Fluoresc (2012) 22:253–259
Stoichiometries and Affinity Constants of 1-Cu2+
Complexes
600
400
200
0
600
400
200
0
In order to understand the binding stochiometry of chemo-
sensor 1-Cu2+ complexes, Job plot experiments were
carried out. Figure 4 plots the emission intensity at
455 nm against molar fraction of chemosensor 1 given a
constant total concentration. Maximum emission intensity
was reached when the molar fraction was 0.5,
corresponding to a 1:1 ratio between chemosensor 1 and
Cu2+. This binding ratio (1:1) for 1-Cu2+ complexes was
also supported by ESI Mass in which a peak at 412 (m/z)
represents the formation of a 1:1 complex. The association
constant Ka was evaluated graphically by plotting 1/ΔF
against 1/[Cu2+] (Fig. 5). The data were linearly fit
according to the Benesi–Hilderbrand equation. The Ka
value, obtained from the slope and intercept of the line,
was found to be 2.75*103 M−1.
0
2
4
6
8
10
Eq of Cu2+
400
500
600
Wavelength (nm)
Fig. 2 Fluorescence response of chemosensor 1 (25 μM) to various
equivalents of Cu2+ in methanol-water (v/v=7:3, 6 mM HEPES, pH
7.0) solutions. The excitation wavelength was 360 nm
To gain a clearer understanding of the structure of
chemosensor 1-Cu2+ complexes, 1H NMR and Infrared (IR)
spectroscopy were employed. Cu2+ is a paramagnetic ion
and can affect the proton signals that are close to a Cu2+
intensity reached a maximum. The quantum yield of that
emission band was 0.267, which is 20-fold that of chemo-
sensor 1, 0.013. These observations indicate that Cu2+ is the
only metal ion that readily binds with chemosensor 1,
significantly enhancing fluorescence and permitting highly
selective detection of Cu2+.
1
binding site. In the H NMR spectra of chemosensor 1,
adding Cu2+ caused the proton (amide NH) signal at
12.4 ppm to almost completely disappear (Fig. 6), the
proton (at pyridine) signals at 7.6 and 8.8 ppm to
disappear, and the intensity of the proton (CH=N) signal
at 9.8 ppm to decrease. Other peaks (protons at pyrene)
remained unchanged. These observations indicated the
binding of Cu2+ with an amide group and pyridine. The
IR spectra were primarily characterized by bands in the
double-bond region. The band 1660 cm−1 was associated
with double-bond (C=O and C=N) absorption in chemo-
sensor 1. Binding of Cu2+ with chemosensor 1 resulted in
a new broad band at 1633 cm−1 in the double-bond
absorption region, due to the amide group in chemosensor 1.
To study the influence of other metal ions on Cu2+
binding with chemosensor 1, this research tested Cu2+
(100 μM) in combination with each of the other metal ions
(100 μM) (Fig. 3). Fluorescence enhancement caused by
the mixture of Cu2+ with most metal ions was similar to
that caused by Cu2+ alone. This observation indicates that
most of the other metal ions did not interfere with the
binding of chemosensor 1 with Cu2+.
600
500
400
300
200
100
160
120
80
40
0
0
Cu(II) Ag(I) Ca(II) Cd(II) Co(II) Fe(II) Fe(III) Hg(II) K(I) Mg(II) Mn(II) Ni(II) Pb(II) Zn(II)
0.0
0.2
0.4
0.6
0.8
1.0
Fig. 3 Fluorescence response of chemosensor 1 (25 μM) to Cu2+
(100 μM) or 100 μM of other metal ions (black bars) and to the
mixture of the other metal ions (100 μM) with 100 μM of Cu2+ (gray
bar portions) in methanol-water (v/v=7:3, 6 mM HEPES, pH 7.0)
solutions
X = [1]/([1]+[Cu2+])
Fig. 4 Job plot of the chemosensor 1-Cu2+ complexes in methanol-
water (v/v=7:3, 6 mM HEPES, pH 7.0) solutions. The total
concentration ([ chemosensor 1 ] + [ Cu2+ ]) was 25μM

J Fluoresc (2012) 22:253–259
257
0.012
0.010
0.008
0.006
0.004
0.002
0.000
The study performed pH titration of chemosensor 1 to
investigate a suitable pH range for Cu2+ sensing. As
depicted in Fig. 8, the emission intensities of metal-free
chemosensor 1 were very low. When pH fell below 2, the
emission intensity increased, due to the protonation on the
amine in the imine bond. After mixing chemosensor 1 with
Cu2+, the emission intensity at 455 nm increased and
reached maximum in the pH range of 6–8. Above pH 8.0,
the emission intensity decreased. This indicates poor
stability of the chemosensor 1-Cu2+ complexes at high pH
values. At pH<4, the emission intensity decreased, due to
the protonation of the amine groups that prevented the
formation of chemosensor 1-Cu2+ complexes.
Intercept = 1.46 ∗10-4
Slope = 5.30 ∗10-8
Ka = 2.75∗103 M-1
0
50000
100000
150000
200000
1 / [Cu2+
]
Fig. 5 Benesi-Hildebrand plot of the chemosensor 1-Cu2+ complexes
in methanol-water (v/v=7:3, 6 mM HEPES, pH 7.0) solutions
The Job plot indicates that the binding ratio for chemo-
sensor 1-Cu2+ complexes was 1:1. Cu2+ was bound to one
nitrogen atom from pyridine and one nitrogen atom from
amide (Fig. 7).
Live Cell Imaging
Chemosensor 1 was further applied for live cell imaging.
For the detection of Cu2+ in live cells, HeLa cells were
Fig. 6 1H NMR spectra of
Chemosensor 1 (5 mM) in the
presence of different amount of
Cu2+ in DMSO-d6
3
4
5
2
1
O
N
N
6
NH
9
8
7
10
11
21
12
13
20
19
22
23
14
15
18
17
1
16
(d)
(c)
(b)
(a)

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J Fluoresc (2012) 22:253–259
Fig. 7 A proposed 1:1 complex
formed between chemosensor 1
and Cu2+
Cu2+
N
N
H
N
N
N
N
Cu2+
O
+
O
1
Weak Fluorescence
Strong Fluorescence
cultured in DMEM supplemented with 10% FBS at 37 °C
and 5% CO2. Cells were plated on 14 mm glass coverslips
and allowed to adhere for 24 h. HeLa cells were treated
with 10 μM CuCl2 for 1 h and washed with PBS for three
times. Then cells were incubated with chemosensor 1
(10 μM) for 30 min and washed with PBS to remove the
remaining sensor. The images of the HeLa cells were
obtained by a fluorescence microscope. Figure 9 shows the
images of HeLa cells with chemosensor 1 after the
treatment of Cu2+. The overlay of fluorescence and bright-
field images reveals that the fluorescence signals are
localized in the intracellular area, indicating a subcellular
distribution of Cu2+ and good cell-membrane permeability
of chemosensor 1.
sensor solution barely affected fluorescence emission. The
optimal pH range for Cu2+ detection by chemosensor 1 is
5~8. This pyrene-based Cu2+ chemosensor also provides an
effective method of Cu2+ sensing in live cell imaging.
Conclusion
This study developed a pyrene-based fluorescent chemosensor
for Cu2+ sensing. The experiment synthesized chemosensor 1
from the reaction of 1-pyrenecarboxaldehyde hydrazone and
picolinoyl chloride to form an amide bond. Fluorescence was
significantly enhanced with chemosensor 1 in the presence of
Cu2+, but adding instead Ag+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+,
Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, or Zn2+ to the chemo-
800
1
1 + Cu2+
600
400
200
0
2
4
6
8
10
12
pH
Fig. 8 Fluorescence intensity (455 nm) of free chemosensor 1
(25 μM)(black squares) and after addition of Cu2+ (100 μM) (blue
circles) in methanol-water (v/v=7:3, 6 mM beffer) solutions as a
function of pH. The excitation wavelength was 360 nm
Fig. 9 Cu2+-treated HeLa cell images. a bright field image; b
fluorescence image; c merged image

J Fluoresc (2012) 22:253–259
259
Acknowledgments We gratefully acknowledge the financial sup-
ports of the National Science Council (ROC) and National Chiao Tung
University.
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