A novel Zn2+ complex as the ratiometric two-photon fluorescent probe for biological Cd2+ detection

Article abstract of DOI:10.1016/j.dyepig.2013.09.013

A novel Zn²⁺ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd²⁺ detection was synthesized. Fluorescence emission spectra of the probe showed a large red-shift (75 nm) and obvious enhancement of fluorescent intensity upon the addition of Cd²⁺. HQZn shows high selectivity for Cd²⁺ over other metal ions, and can eliminate the interference of Zn²⁺ during Cd²⁺ detection. Maldi-TOF MS spectra indicated that the response of HQZn to Cd²⁺ was caused by central metal displacement. Cell cytotoxicity and bio-imaging studies revealed that HQZn was cell-permeable and it could be used to detect intracellular Cd²⁺ with low cytotoxicity under two-photon excitation.

Full text of DOI:10.1016/j.dyepig.2013.09.013

Dyes and Pigments 101 (2014) 30e37
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A novel Zn2þ complex as the ratiometric two-photon fluorescent
probe for biological Cd2þ detection
,
a
a
Weipeng Ye a, Shuxing Wang a, Xiangming Meng a , Yan Feng , Hongting Sheng ,
*
Zonglong Shao a, Manzhou Zhu a , Qingxiang Guo
,
b
*
a School of Chemistry and Chemical Engineering, Anhui University, 230601 Hefei, PR China
b Department of Chemistry, University of Science and Technology, Hefei 230026, PR China
a r t i c l e i n f o
a b s t r a c t
A novel Zn2þ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd2þ detection was
synthesized. Fluorescence emission spectra of the probe showed a large red-shift (75 nm) and obvious
enhancement of fluorescent intensity upon the addition of Cd2þ. HQZn shows high selectivity for Cd2þ
over other metal ions, and can eliminate the interference of Zn2þ during Cd2þ detection. Maldi-TOF MS
spectra indicated that the response of HQZn to Cd2þ was caused by central metal displacement. Cell
cytotoxicity and bio-imaging studies revealed that HQZn was cell-permeable and it could be used to
detect intracellular Cd2þ with low cytotoxicity under two-photon excitation.
Article history:
Received 14 June 2013
Received in revised form
30 August 2013
Accepted 10 September 2013
Available online 27 September 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Ratiometic
Two-photon
Fluorescent probe
Zn2þ complex
Cd2þ
Bio-imaging
1. Introduction
increased penetration depth, localized excitation and prolonged
observation time [18e21]. Many two-photon fluorescent probes
Cadmium, as one of the most toxic metal pollutant, can accu-
mulate in the human body for more than 10 years and cause many
serious diseases, such as renal dysfunction, calcium metabolism
disorders, prostate cancer, etc. [1,2]. Therefore, detection and
quantification of Cd2þ in vitro and in vivo is highly needed.
have been developed for detecting various metal ions, while two-
photon probes for Cd2þ (especially ratiometric) have rarely been
reported [22,23].
Although Zn2þ and Cd2þ belong to the same group, they play
totally different roles in biochemical processes. Cd2þ can replace
Zn2þ in many zinc enzymes, thereby impairing their catalytic ac-
tivities [24]. So far, most of the reported fluorescent probes for Cd2þ
suffered from the interference from Zn2þ, which can be ascribed to
highly similar chemical properties of Zn2þ and Cd2þ [25e30].
Therefore, it is still a challenging task to develop highly selective
fluorescent probes for Cd2þ, especially avoiding the interference of
Zn2þ in detection. Previous reported results indicated that Cd2þ can
displace Zn2þ in its complex and give different emission fluores-
cence due to their different coordination mode [31]. Thus we ask an
interesting question whether or not we can detect Cd2þ based on a
Zn2þ complex by central metal displacement. If our goal can be
achieved, we will eliminate the Zn2þ interference during Cd2þ
detection. Inspired by this idea and our recent success in devel-
oping two-photon probes for Cd2þ and Zn2þ based on 6-substituted
quinoline skeletons [32e34], we designed a 6-substituted quino-
line Zn2þ complex (HQZn, Scheme 1) as the ratiometric two-
photon fluorescent probe for Cd2þ. Maldi-TOF MS spectra was
Fluorescent probes have been regarded as the most powerful
tools for monitoring metal ions in biological and environmental
samples for their high selectivity and sensitivity [3e6]. Many
fluorescent probes for Cd2þ have been reported, and most of them
were “off-on” type [7e13]. Detecting Cd2þ depended on the change
of the emission at only one wavelength, which may cause difficulty
in quantitative determination and bio-imaging due to the back-
ground interference (pH, polarity, temperature, and so forth) [14e
17]. Ratiometric probes can overcome these limitations because of
their self-calibration by measuring the ratio of emission at two
different wavelengths. Recently, two-photon microscopy (TPM) has
been evolved into a widely used tool in biomedical research for its
evident advantages over one-photon microscopy (OPM), including
* Corresponding authors. Tel./fax: þ86 551 63861467.
E-mail addresses: mengxm@ahu.edu.cn (X. Meng), zmz@ahu.edu.cn (M. Zhu).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.09.013

W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
31
Scheme 1. The design of HQZn for Cd2þ detection.
used to reveal that the response of HQZn to Cd2þ was caused by
central metal displacement. Meanwhile, cell cytotoxicity and bio-
imaging studies indicated that HQZn was cell-permeable and it
could be used to detect intracellular Cd2þ with low cytotoxicity
under two-photon excitation.
spectrophotometer. Fluorescence spectra were recorded on a
HITACHIF-2500 spectrometer. MS spectra were recorded on a
Bruker autoflex III MALDI-TOF mass spectrometer. Cells imaging
was carried out on a Zeiss LSM 510 Meta NLO confocal microscope.
2.2. Synthesis procedure
2. Experimental section
2.2.1. 6-bromo-2-methylquinoline (1)
A mixture of 4-bromobenzenamine (14.59 g, 84.8 mmol) and
HCl (6 N, 60 mL) was heated to 100 ꢀC, then crotonaldehyde (11.9 g,
170 mmol) was added slowly. The resulting mixture was refluxed
until TLC shows no raw material exists. After cooling to room
temperature, 200 mL H2O was added and the mixture was
extracted with EtOAc (100 mL ꢁ 2) to remove the unreacted cro-
tonaldehyde. The aqueous phase was neutralized with ammonia
water and then extracted with EtOAc (50 mL ꢁ 2). The organic
phase was dried over anhydrous Na2SO4 and evaporated to give
crude residue. The crude product was recrystallized in EtOAc/PE to
give 13.46 g of 1 (60.64 mmol, 75.6%). 1H NMR (400 MHz, CDCl3,
2.1. Materials and physical measurements
All reagents were commercially purchased, and the solvents
were used after appropriate distillation or purification. The solu-
tions of metal ions were prepared from chloride salts. HEPES buffer
solutions (20 mM, pH 7.4) were prepared in water. 1H NMR spectra
were recorded on Bruker-400 MHz spectrometers and 13C NMR
spectra were recorded on 100 MHz spectrometers. All solvents used
in the test were chromatographically pure. UVevis absorption
spectra were recorded on
a
Tech-comp UV 1000
ppm):
d
2.73 (3H, s), 7.29e7.31 (1H, d, J ¼ 8.4 Hz), 7.73e7.75 (1H, d,
J ¼ 9.0 Hz), 7.87e7.96 (3H, m). 13C NMR (100 MHz, CDCl3, ppm):
d
25.18, 119.57, 122.91, 127.64, 129.54, 130.12, 133.03, 135.49, 146.05,
159.45.
2.2.2. (E)-6-(4-methoxystyryl)-2-methylquinoline (2)
A mixture of 1 (6.03 g, 27.15 mmol), 4-methoxystyrene (4.37 g,
32.58 mmol), PdCl2(PPh3)2 (77 mg, 0.97 mmol), K2CO3 (11 g,
79.7 mmol) and DMF (25 mL) was heated at 160 ꢀC for 24 h [35].
After cooling to room temperature, the mixture was filtered to
remove salts, and then 100 mL H2O was added. The result mixture
was extracted by DCM (50 mL ꢁ 3). The organic phase was dried
over anhydrous Na2SO4 and evaporated to give crude residue. The
crude product was recrystallized in EtOAc/PE to give 6.77 g of 2
(24.63 mmol, 90.72%). 1H NMR (400 MHz, CDCl3, ppm):
d 2.75 (3H,
s), 3.84 (3H, s), 6.91e6.93 (2H, d, J ¼ 8.6 Hz), 7.08e7.21 (2H, q,
J ¼ 16.3 Hz), 7.25e7.27 (1H, d, J ¼ 8.3 Hz), 7.48e7.50 (2H, d,
J ¼ 8.6 Hz), 7.73 (1H, s), 7.91e7.93 (1H, d, J ¼ 8.9 Hz), 8.01e8.03 (2H,
d, J ¼ 8.5 Hz). 13C NMR (100 MHz, CDCl3, ppm):
d 25.11, 55.35,
Scheme 2. Synthetic route of HQZn.
Fig. 1. X-ray crystal structure for HQZn.

32
W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
Table 1
NMR (400 MHz, DMSO-d6, ppm): d 3.80 (3H), 6.92e6.94 (3H), 7.27e
7.49 (2H), 7.60e7.63 (2H), 7.71e8.05 (3H), 8.07e8.12 (2H), 8.18e
8.31 (2H), 8.49e8.56 (1H), 10.95e15.28 (1H).
Crystallographic data for HQZn.
Compound
HQZn
C27H27Cl2N5O2Zn
Chemical formula
Formula mass
Crystal system
589.81
Monoclinic
13.8914(5)
11.0111(5)
20.4872(7)
90
105.874(4)
90
3014.2(2)
291(2)
P 21/n
2.2.5. Zinc(Ⅱ) complex of HQ (HQZn)
HQ (120 mg, 0.3 mmol) and ZnCl2 (0.04 g, 0.3 mmol) were
dissolved in 4 mL DMF and stirred for 2 h at room temperature,
then 2 mL Et2O was added. Orange acicular single crystals were
obtained at room temperature after a few days. 1H NMR (400 MHz,
ꢀ
a/A
ꢀ
b/A
ꢀ
c/A
ꢀ
ꢀ
a
/
b
/
DMSO-d6, ppm):
d 3.80 (3H), 6.97e7.11 (3H), 7.27e7.34 (1H), 7.35e
ꢀ
g
/
3
ꢀ
7.45 (1H), 7.7e8.1 (4H), 8.15e8.37 (2H), 8.45e8.85 (2H), 12.80e
15.95 (1H).
Unit cell volume/A
Temperature/K
Space group
Z
Number of reflections measured
Number of independent reflections
Rint
4
2.3. X-ray crystallography
16,466
4815
0.0407
0.0560
0.1545
0.0796
0.1702
X-ray diffraction data of HQZn single crystals were collected on
a Siemens Smart 1000 CCD diffractometer. The determination of
Final R1 values (I > 2s(I))
Final wR(F2) values (I > 2
Final R1 values (all data)
s(I))
unit cell parameters and data collections were performed with Mo
ꢀ
Final wR(F2) values (all data)
Ka
radiation (
l
¼ 0.71073 A). Unit cell dimensions were obtained
with least-squares refinements, and all structures were solved by
direct methods using SHELXS-97 [36]. The other non-hydrogen
atoms were located in successive difference Fourier syntheses.
The final refinement was performed by using full-matrix least-
squares methods with anisotropic thermal parameters for non-
hydrogen atoms on F2. The hydrogen atoms were added theoreti-
cally and rode on the concerned atoms.
114.23, 122.39, 125.19, 125.79, 126.81, 127.39, 127.87, 128.59, 129.36,
129.90, 135.26, 136.35, 147.07, 158.43, 159.54.
2.2.3. (E)-6-(4-methoxystyryl)quinoline-2-carbaldehyde (3)
A solution of 2 (2.00 g, 7.27 mmol) in dioxane (20 mL) was
heated to 60 ꢀC, and SeO2 (8.00 mmol, 0.889 g) was added to this
solution. The reaction temperature was kept at 80 ꢀC for 2.5 h, then
the mixture was cooled to room temperature. Precipitates were
filtered off and washed with dioxane (5 mL ꢁ 2). The organic phase
was combined and concentrated to give a crude product. The crude
material was purified by column chromatography (DCM as the
eluent) to give 1.818 g of 3 (6.61 mmol, 90.9%). 1H NMR (400 MHz,
2.4. Preparation of fluorescent titration
Inorganic salt was dissolved in distilled water to afford 10 mM
aqueous solution. The 1 mM stock solution of HQZn was prepared
in absolute methanol. All the measurements were carried out ac-
cording to the following procedure. To 10 mL volumetric flask
CDCl3, ppm):
d
3.85 (3H, s), 6.92e6.94 (2H, d, J ¼ 8.6 Hz), 7.11e7.30
(2H, dd, J ¼ 16.1 Hz), 7.50e7.52 (2H, d, J ¼ 8.6 Hz), 7.82 (1H, s), 7.99e
8.05 (2H, dd, J ¼ 16.4 Hz), 8.18e8.25 (2H, dd, J ¼ 18.1 Hz), 10.21 (1H,
containing 250
mL of the solution of HQZn, different amounts
(25 Le1000 L) of metal ions were added directly with micropi-
m
m
s). 13C NMR (100 MHz, CDCl3, ppm):
d 55.37, 114.33, 117.88, 125.11,
pette, then diluted with buffered (pH 7.4, 20 mM HEPES) solution.
For the Job’s plot experiment, HQZn (1 mM) in absolute methanol
and CdCl2 (1 mM) in distilled water were prepared as stock solu-
tions. The methanolewater solution (3:7, v/v, 20 mM HEPES buffer,
pH ¼ 7.4) was prepared. The concentrations of HQZn and Cd2þ
solution were varied, but the total volume was fixed at 10 mL and
125.15, 128.19, 128.30, 129.41, 130.54, 130.64, 131.37, 136.97, 138.71,
147.44, 151.91, 159.97, 193.51.
2.2.4. (E)-6-(4-methoxystyryl)-2-(2-(pyridin-2-yl)hydrazono)
quinoline (HQ)
the total concentrations of HQZn and Cd2þ are 25
mM. After the
A mixture of 3 (0.5 g, 1.73 mmol) and 2-hydrazinylpyridine
(0.22 g, 2.07 mmol) in ethanol (10 mL) was heated to 60 ꢀC. The
reaction temperature was kept at 60 ꢀC for 0.5 h. After cooling to
room temperature, precipitates were collected by filtration and
recrystallized in EtOH to give 0.56 g of HQ (1.42 mmol, 80%). 1H
mixture was shaken, the maximum of fluorescence intensity was
recorded. Fluorescence measurements were carried out with exci-
tation and emission slit width of 10 and 10 nm and PMT Voltage
and excitation wavelength was 400 V and 400 nm, respectively.
Fig. 2. (a) UVevis spectra of HQ (25 m
M) upon the titration of Zn2þ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2 equiv) in the methanolewater solution (3:7, v/v, 20 mM HEPES
buffer, pH ¼ 7.4). (b) UVevis spectra of HQZn (25
m
M) upon the titration of Cd2þ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2 equiv) in the methanolewater solution (3:7, v/v,
20 mM HEPES buffer, pH ¼ 7.4).

W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
33
Fig. 3. (a) Fluorescence responses (lex ¼ 360 nm) of HQ (25
m
M) upon the titration of Zn2þ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2 equiv) in the methanolewater solution
(3:7, v/v, 20 mM HEPES buffer, pH ¼ 7.4). (b) Ratiometric calibration curve (F485 nm/F460 nm) as a function of the Zn2þ concentration. (c) Fluorescence responses (lex ¼ 400 nm) of
HQZn (25
m
M) upon the titration of Cd2þ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2 equiv) in the methanolewater solution (3:7, v/v, 20 mM HEPES buffer, pH ¼ 7.4). Inset:
Digital photographs of HQZn and HQZn in presence of equivalent amount of Cd2þ under a hand-held UVevis (365 nm) lamp. (d) Ratiometric calibration curve (F560 nm/F485 nm) as a
function of the Cd2þ concentration.
2.5. Measurement of two-photon absorption cross section (
d
)
Two-photon absorption cross sections were measured using two-
photon-induced fluorescence measurement technique. The two-
Two-photon excitation fluorescence (TPEF) spectra were
measured using femtosecond laser pulse and Ti: sapphire system
(680e1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source.
All measurements were carried out in air at room temperature.
photon absorption cross sections (d) were determined in CH3OH
by comparing their TPEF to that of fluorescein according to the
literature [37].
2.6. Cytotoxicity assays
MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide)
assay was performed as previously reported to test the cytotoxic
effect of the probe in cells [32]. HeLa cells were passed and plated to
ca. 70% confluence in 96-well plates 24 h before treatment. Prior to
HQZn treatment, DMEM (Dulbecco’s Modified Eagle Medium) with
10% FCS (Fetal Calf Serum) was removed and replaced with fresh
DMEM, and aliquots of HQZn stock solutions (5 mM DMSO) were
added to obtain final concentrations of 10, 30, and 50
tively. The treated cells were incubated for 24 h at 37 ꢀC under 5%
CO2. Subsequently, cells were treated with 5 mg/mL MTT (40 L/
well) and incubated for an additional 4 h (37 ꢀC, 5% CO2). Then the
cells were dissolved in DMSO (150 L/well), and the absorbance at
mM respec-
m
m
570 nm was recorded. The cell viability (%) was calculated accord-
ing to the following equation: Cell viability% ¼ OD570(sample)/
OD570(control) ꢁ 100, where OD570(sample) represents the optical
density of the wells treated with various concentration of HQZn
and OD570(control) represents that of the wells treated with DMEM
containing 10% FCS. The percent of cell survival values is relative to
untreated control cells.
Fig. 4. Job’s plot of HQZn and Cd2þ
centrations of HQZn and Cd2þ are 25
temperature in the methanolewater solution (3:7, v/v, 20 mM HEPES buffer, pH ¼ 7.4).
(
lex ¼ 400 nm, lem ¼ 560 nm). The total con-
M. The experiments were measured at room
m

34
W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
Fig. 7. Ratio (F560 nm/F485 nm) of HQZn upon addition of various metal ions (1e15: Naþ
,
Agþ, Mn2þ, Cu2þ, Hg2þ, Pb2þ, Mg2þ, Ca2þ, Fe2þ, Co2þ, Ni2þ, Cr3þ, Fe3þ, Zn2þ, Cd2þ) in
the methanolewater solution (3:7, v/v, 20 mM HEPES buffer, pH ¼ 7.4, lex ¼ 400 nm).
The black bars represent the emission of HQZn in the presence of 1 equiv of different
metal cations to HQZn. The red bars represent the change in integrated emission that
occurs upon subsequent addition of 1 equiv of Cd2þ to the solution (lex ¼ 400 nm). (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article).
CO2 and 95% air. Cytotoxicity assays show that HQZn is safe enough
for two-photon bio-imaging at low concentrations, so that the cells
were incubated with 15
washed once and bathed in DMEM containing no FCS prior to im-
aging. Then 30
M Cd2þ was added in the growth medium for 0.5 h
m
M HQZn at 37 ꢀC under 5% CO2 for 30 min,
m
at 37 ꢀC, washed 3 times with PBS buffer. Cells imaging was carried
out on a confocal microscope (Zeiss LSM 510 Meta NLO). Two-
photon fluorescence microscopy images of labeled cells were ob-
tained by exciting the probe with a mode-locked titanium-sapphire
laser source set at wavelength 730 nm.
Fig. 5. (a) MALDI-TOF MS spectrum of HQZn (DCTB as matrix). (b) MALDI-TOF MS
spectrum of HQZneCd2D complex (DCTB as matrix).
2.7. Cell culture and two-photon fluorescence microscopy imaging
3. Results and discussion
3.1. Structure of HQZn and HQZneCd2þ
For two-photon bio-imaging, HeLa cells were cultured in DMEM
supplemented with 10% FCS, penicillin (100
mg/mL), and strepto-
mycin (100
m
g/mL) at 37 ꢀC in a humidified atmosphere with 5%
As shown in Scheme 2, the probe HQZn was synthesized via five
steps from readily available 4-bromoaniline with overall yield of
Fig. 6. Two-photon absorption cross sections of HQZn and HQZneCd2D in CH3OH
under different exciting wavelengths of identical energy of 0.300
w
Fig. 8. Ratio (F560 nm/F485 nm) of HQZn and HQZneCd2D at various pH values in the
methanolewater solution (3:7, v/v, 20 mM HEPES buffer, lex ¼ 400 nm).
(c ¼ 1.0 ꢁ 10ꢂ3 mol/L).

W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
35
of the complex of HQZn with a molar ratio of 1:1 (HQ/Zn2þ). The
red-shift of UVevis and fluorescence spectra indicates that the ni-
trogen atom of the quinoline platform is coordinated with Zn2þ to
cause an enhanced ICT process from donor (methoxy group) to
acceptor (quinoline) [9,33].
Interestingly, upon the addition of Cd2þ to the solution of HQZn,
the maximum absorption spectra were further shifted to 430 nm
with a clear isosbestic point at 416 nm (Fig. 2(b)). The emission
spectra showed a larger red-shift of 75 nm from 485 nm to 560 nm
with an isoemissive point at 504 nm (Fig. 3(c)). Meanwhile, the
color of the fluorescence turned from green to yellow (Fig. 3(c)
inset). The ratio of emission intensity (F560 nm/F485 nm) increased
linearly from 0.5 to 4.0 with Cd2þ addition and was saturated up to
a molar ratio (HQZn/Cd2þ) of 1:1 (Fig. 3(d)). The further red-shift of
the spectra of HQZn indicated that the core metal ion (Zn2þ) of the
complex was replaced by Cd2þ, which further enhanced the ICT
process. Job’s plot analysis (Fig. 4) also confirmed the displacement.
Fig. 9. Cytotoxicity data of HQZn (HeLa cells incubated for 24 h).
The linear enhancement of the ratio of emission intensity (F560 nm
/
F485 nm) of HQZn upon the addition of Cd2þ suggested that HQZn
could be served as an efficient ratiometric fluorescent probe for
40%. The structure of HQZn was confirmed by 1H NMR and MS
spectra. The single crystal of HQZn grew from anhydrous Et2O/DMF
solution. The crystal structure diagrams are shown in Fig. 1. The
details of the crystallographic data are listed in Table 1. As shown in
Fig. 1, the crystal system of HQZn is monoclinic and Zn2þ is coor-
dinated to three nitrogen atoms of HQ and two chloride ions
forming a distorted square pyramidal.
Cd2þ
.
Another solid evidence for displacement reaction came from the
Maldi-TOF MS spectrum. As shown in Fig. 5, HQZn has a peak at m/
z ¼ 481.07 ([HQ þ ZnCl]þ), upon the addition of Cd2þ, a peak at m/
z ¼ 529.07 ([HQ þ CdCl]þ) appeared along with the disappearance
of the peak at m/z ¼ 481.07. The observations confirmed that Zn2þ
of HQZn was completely displaced by Cd2þ to form the corre-
sponding Cd2þ complex via the central metal displacement.
According to the reported method [38,39], the dissociation
3.2. UVevis and fluorescence spectra studies
constants (Kd) of HQ for Zn2þ and Cd2þ were calculated to be 9.6
mM
and 21 pM, respectively. This confirmed that Cd2þ exhibited higher
affinity for HQ and can displace the Zn2þ of HQZn to form a more
stable complex.
All spectroscopic measurements were carried out under simu-
lated physiological conditions (20 mM HEPES buffer, pH ¼ 7.4). As
shown in Fig. 2, the UVevis absorption spectra of HQ exhibited a
maximum absorption at 355 nm in methanolewater solution.
Upon increasing the amount of Zn2þ, the maximum absorption was
gradually shifted to 394 nm along with the enhancement of in-
tensity (Fig. 2(a)). HQ exhibited weak fluorescence emission at
460 nm up excitation at 400 nm (Fig. 3(a)). When saturated with
Zn2þ, emission spectra showed a red-shift of 25 nm from 460 to
485 nm with a slight enhancement of intensity. The ratio of emis-
sion intensity (F485 nm/F465 nm) increased very slowly from 0.86 to
1.15 with Zn2þ addition (Fig. 3(b)). These confirmed the formation
3.3. Two-photon absorption studies
Considering the application of HQZn in living system, we further
determine the two-photon absorption cross section of HQZn and its
corresponding Cd2þ complex using the two-photon induced fluo-
rescence measurement technique. As shown in Fig. 6, the
maximum two-photon absorption cross section (dmax) value of
Fig. 10. (a) Bright-field image of Hela cells. (b) TP image of Hela cells disposed with 15
wavelength from 470 nm to 500 nm). (c) Emission wavelength from 545 nm to 580 nm. (d) The overlay of (a) (b) and (c). (e) Bright-field image of Hela cells. (f) TP image following a
m
M HQZn after 30 min of incubation, washed by PBS buffer. lex ¼ 730 nm (emission
30 min treatment with CdCl2 (30 mM). Emission wavelength from 470 nm to 500 nm. (g) Emission wavelength from 545 nm to 580 nm. (h) The overlay of (e) (f) and (g).

36
W. Ye et al. / Dyes and Pigments 101 (2014) 30e37
HQZn is 48 GM at 730 nm. Upon addition of 1.2 equiv. Cd2þ, the
dmax value increases greatly to 85 GM at 730 nm. The obvious
enhancement of two-photon excitation fluorescence makes HQZn
a potential two-photon probe for monitoring Cd2þ flux in living
systems.
when the central metal ion was displaced by Cd2þ. Importantly,
HQZn shows high selectivity for Cd2þ over other metal ions, and
can eliminate the interference of Zn2þ during Cd2þ detection.
Finally, in vivo two-photon microscopy imaging and cell cytotox-
icity demonstrated that the new probe is cell-permeable and can be
used to monitor the Cd2þ flux in living cells. This work may provide
3.4. Ions selectivity and pH stability
a new strategy to design probes for discriminating Cd2þ and Zn2þ
.
Metal ion selectivity studies were performed in HEPES buffer. As
shown in Fig. 7 (black bars), Naþ, Kþ, Ca2þ, and Mg2þ, which are
abundant in living cells, exerted a negligible effect on the fluores-
cence of HQZn even at higher concentrations (40 times of the
Acknowledgments
This work was supported by NSFC (21102002, 21372005,
21102001, and 21272223), Natural Science Foundation of Education
Department of Anhui Province (KJ2011A018), 211 Project of Anhui
University and Doctor Research Start-up Fund of Anhui University
for supporting the research.
HQZn). Transition-metal ions, including Agþ, Mn2þ, Cu2þ, Hg2þ
,
Pb2þ, Fe2þ, Co2þ, Ni2þ, Cr3þ and Fe3þ did not cause observable
change of the fluorescence of HQZn. Competitive experiments were
also carried out in presence of 1.0 equiv of Cd2þ mixed with
1.0 equiv of other metal ions. As shown in Fig. 7 (red bars), both
alkali and transition-metal ions exert little effects on the ratio-
metric Cd2þ detecting signal (F560 nm/F485 nm) of HQZn. It’s impor-
tant to point out that Zn2þ, which often interferes the Cd2þ
detection, causes non-change of the fluorescent spectra of HQZn as
expected. These results suggest that HQZn has excellent selectivity
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2013.09.013
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