Ratiometric and selective two-photon fluorescent probe based on PET-ICT for imaging Zn2+ in living cells and tissues

Article abstract of DOI:10.1016/j.cclet.2013.10.005

A two-photon fluorescent probe TPZn was developed for specific ratiometric imaging Zn²⁺ in living cells and tissues. Significant ratiometric fluorescence change was based on photoinduced electron transfer and intramolecular charge transfer. The synthetic method of TPZn was simple. It was successfully used to selectively image Zn²⁺ based on the higher binding affinity for Zn²⁺ than for Cd²⁺. TPZn was easily loaded into the living cell and tissues with high membrane permeability in a complex biological environment. TPZn could clearly visualize endogenous Zn ²⁺ by TP ratiometric imaging in hippocampal slices at a depth of 120 μm. Thus, TPZn is a useful tool to image of Zn²⁺ in living cells and tissues without interference from Cd²⁺.

Full text of DOI:10.1016/j.cclet.2013.10.005

Chinese Chemical Letters 25 (2014) 93–98
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Ratiometric and selective two-photon fluorescent probe based
on PET-ICT for imaging Zn2+ in living cells and tissues
*
Shang Wu , Ya-Jun Wei, Yan-Bin Wang, Qiong Su, Lan Wu, Hong Zhang,
Fen-Ping Yin, Pei-Lin Yuan
College of Chemical Engineering, Northwest University for Nationalities, Lanzhou 730030, China
A R T I C L E I N F O
A B S T R A C T
A two-photon fluorescent probe TPZn was developed for specific ratiometric imaging Zn2+ in living cells
and tissues. Significant ratiometric fluorescence change was based on photoinduced electron transfer
and intramolecular charge transfer. The synthetic method of TPZn was simple. It was successfully used to
selectively image Zn2+ based on the higher binding affinity for Zn2+ than for Cd2+. TPZn was easily loaded
into the living cell and tissues with high membrane permeability in a complex biological environment.
TPZn could clearly visualize endogenous Zn2+ by TP ratiometric imaging in hippocampal slices at a depth
Article history:
Received 16 July 2013
Received in revised form 15 September 2013
Accepted 17 September 2013
Available online 19 November 2013
Keywords:
of 120
from Cd2+
m
m. Thus, TPZn is a useful tool to image of Zn2+ in living cells and tissues without interference
Two-photon probe
Ratiometric fluorescent imaging
Zinc
.
ß 2013 Shang Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Living cell
Living tissue
1. Introduction
tissues, for example, AZn1, AZn2 and Zinbo-5 [10]. These probes
have significantly enriched our knowledge about the role Zn2+
Zinc is of great interest in the field of neurobiology [1,2]. Recent
studies indicate that cellular Zn2+ levels are tightly regulated [3].
There is a pool of Zn2+ that is loosely bound in the vesicles of
hippocampal CA3 neurons [2]. It is also known that disorder of Zn2+
metabolism is closely associated with many severe neurological
diseases, including Alzheimer’s disease (AD) [4], cerebral ischemia,
plays in many biological processes.
Herein, we report a novel two-photon ratiometric probe for
Zn2+ ions (TPZn, Scheme 1) in living cells and tissues, which is
based on photoinduced electron transfer (PET) and intramolecular
charge transfer (ICT). TPZn is derived from 1,4-dicyano-2,5-
bis(styryl)benzene as the fluorophore based on a donor–accep-
tor–donor structure possessing a large two-photon cross section
and N,N-bis(pyridine-2-ylmethyl)benzenamine as a Zn2+ receptor
(ICT donor). We also note that TPZn successfully discriminates Zn2+
from Cd2+, which typically interferes in the Zn2+ detection.
and epilepsy [5]. To understand the biological functions of Zn2+
many fluorescent sensors have been reported [6].
,
Most of the reported Zn2+ probes are based on one-photon
excitation [7]. In these works, quinolin [7], pyridinehydrazone [8]
and so on were used as fluorophores for the detection of Zn2+. They
are only used in the photography of cells, as the light penetration is
limited and highly localized photography images in deep tissue are
difficult to obtain. To visualize the biological activity deep inside
2. Experimental
TPZn was synthesized according to the general route by Witting
reaction of 1,4-bis(diethylphosphorylmethyl)-2,5-dicyanoben-
zene with 4-(bis(pyridine-2-ylmethyl)amino)-benzaldehyde in
35% yield (Scheme 1).
All the solvents were of analytical grade. The solutions of metal
ions were prepared by dissolving Mn(NO3)2, Pb(NO3)2, Co(N-
O3)2Á6H2O, Zn(NO3)2Á6H2O, Ca(NO3)2Á4H2O, NaNO3, Cu(NO3)2Á3H2O,
3H2O, Ni(NO3)2Á6H2O, KNO3, Cd(NO3)2Á2H2O, Hg(NO3)2Á1/2H2O,
Cr(NO3)3Á9H2O, Mg(NO3)2Á6H2O, FeCl2Á4H2O, and BaCl2Á2H2O in
distilled water. 1H NMR and 13C NMR spectra were recorded on a
VARIAN INOVA-400 spectrometer with chemical shifts reported as
ppm (in CDCl3, TMS as internal standard). Mass spectrometric data
living tissue (>80
mm) without the interference of surface
preparation artifacts [9], it is crucial to use two-photon microscopy
(TPM). The technique, which employs two-photons of lower-
energy for the excitation, has the advantages of an increased
penetration depth,
a localized excitation, and a prolonged
observation time [9]. Recently, Cho et al. reported some two-
photon Zn2+-selective probes to be used in the imaging of cells and
*
Corresponding author.
E-mail address: chwush84@163.com (S. Wu).
1001-8417/$ – see front matter ß 2013 Shang Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.10.005

94
S. Wu et al. / Chinese Chemical Letters 25 (2014) 93–98
CN
CN
N
O
OEt
N
N
N
N
N
Cl
N
N
N
N
N
N
NC
P
N
EtO
O
NC
TPZn
1
2
3
4
Scheme 1. The synthesis of TPZn.
were obtained on an HP1100LC/MSD MS spectrometer and an LC/Q-
Tof MS spectrometer. Fluorescence measurements were performed
on a PTI-700 Felix and Time-Master system, and the slit width was
3 nm for both excitation and emission. Absorption spectra were
measured on Lambda 35 UV/vis spectrophotometer. All pH
measurements were made with a Model PHS-3C meter. The
7.70–7.81 (t, 4H, J = 7.2 Hz), 7.52 (s, 1H), 7.40–7.49 (d, 2H,
J = 8.0 Hz), 7.29–7.34 (d, 4H, J = 4.8 Hz), 7.16–7.21 (d, 4H,
J = 16.0 Hz), 7.09–7.14 (d, 4H, J = 16.0 Hz), 6.99–7.09 (t,
J = 7.2 Hz, 4H), 6.72–6.79 (d, J = 8.6 Hz, 2H), 4.92 (s, 8H). 13C
NMR (100 MHz, CDCl3, Me4Si):
d 158.24, 149.98, 149.31, 138.78,
137.14, 134.46, 129.21, 129.06, 125.03, 122.45, 120.94, 117.90,
117.14, 114.38, 112.87, 77.51, 77.20, 76.88, 57.39. TOF-MS calcd.
for C48H40N8 [M+2H]+: 728.3376, found: 728.3402.
fluorescence quantum yield (
standardmethods onair-equilibratedsamplesatroomtemperature.
Quinine bisulfate in 0.05 mol/L H2SO4 = 0.546) was used as a
reference. The TPA cross sections ( TPA) of TPZn were determined
F) of TPZn was measured using
(F
d
3. Results and discussion
under various conditions by the two-photon-induced fluorescence
method by using fluorescein (10À4 mol/L in 0.1 mol/L NaOH) as the
reference.
Compound 4 was synthesized according to our previous work
[11].
In the TPZn molecule, DPA was used not only as the receptor for
Zn2+, but also as the donor in a push–pull electronic system. The
absorption spectrum of TPZn in CH3CN–water solution is
characterized by a very intense band centered at 450 nm, which
is responsible for the orange-yellow color of the solution.
Compound 2: To a solution of 2-chloromethylprydine (1.524 g,
12 mmol) in H2O (0.5 mL), aniline (0.558 g, 6 mmol), 5 mol/L NaOH
(3 mL), and hexadecytrimethylammonium chloride (20 mg) were
added under N2 protection. The mixture was stirred vigorously for
24 h at room temperature. The mixture was extracted with CH2Cl2,
and the extract was washed with H2O and dried with MgSO4. After
evaporation of the solvent, the desired product 2 (1.262 g) was
obtained as a beige solid in 76% yield via column chromatography
Fluorescence of TPZn was quenched (
F
= 0.09), because PET
ab showed a 50 nm
occurred. When Zn2+ was added gradually, the
l
blue shift from 450 nm to 400 nm with an isosbestic point at
425 nm. The PET process was restrained, and there are blue shift in
the absorption and fluorescence spectra, because the electron-
donating ability of the nitrogen atom at the N,N-bis(pyridin-2-
ylmethyl) aniline conjugated onto the fluorophore was reduced,
which is an ICT process.
(silica, CH2Cl2/AcOEt, 4/1, v/v). 1H NMR (400 MHz, CDCl3):
d 4.83 (s,
4H), 6.71–6.73 (m, 3H, J = 20 Hz), 7.17–7.21 (m, 4H, J = 12 Hz),
7.27–7,28 (d, 2H, J = 8 Hz), 7.62 (t, 2H, J = 16 Hz), 8.60–8.69 (d, 2H,
d 57.37, 112.55, 117.29,
120.86, 122.12, 129.39, 136.93, 148.24, 149.81, 158.91. TOF-MS
calcd. for C18H17N3 [M+H]+: 276.1501, found: 276.1506.
Compound 3: POCl3 (1 mL, 17 mmol) was added into a solution
of DMF (2 mL, 26 mmol) in portions over 0.5 h and cooled in an ice
bath. Then, the solution was stirred for 0.5 h. Compound 2 (0.600 g,
2.18 mmol) in DMF (1 mL) was added in portions over 20 min. The
mixture was heated for 3 h at 90 8C and stirred into H2O (5 mL), and
then neutralized to pH 6–8 with K2CO3. The mixture was extracted
with CH2Cl2, and dried with MgSO4. Via column chromatography
(silica, petroleum/acetone, 5/3, v/v), the desired product (0.263 g)
was obtained as a yellow oil in 40% yield. 1H NMR (400 MHz,
Free TPZn exhibited lem at 586 nm with a quantum yield of
0.09. Upon addition of Zn2+, the lem underwent a blue shift from
586 nm to 535 nm with a quantum yield of 0.43, and the
fluorescence color changed from orange-red to green. Other metal
ions had no such fluorescence response. Cd2+, the most common
interfering ion, only slightly changed the fluorescence intensity
J = 4 Hz). 13C NMR (100 MHz, CDCl3):
with a smaller wavelength shift (15 nm,
Upon gradual addition of Zn2+ to
F
Cd = 0.15 Fig. 1a and b).
a
solution of TPZn, the
fluorescence intensity increased linearly with the Zn2+ concentra-
tion in the range of 0–2.0 mmol/L (Fig. 1a and c).
From the fluorimetric experiments, we realized that TPZn
would have higher binding affinity for Zn2+ than for Cd2+. As
expected, the addition of Zn2+ into TPZn-Cd solution resulted in
quick fluorescence changes (less than 1 min). The lem of TPZn-Cd
CDCl3):
J = 16 Hz), 7.66–7.72 (m, 4H, J = 24 Hz), 8.61–8.71 (d, 2H, J = 4 Hz),
9.71 (s, 1H). 13C NMR (100 MHz, CDCl3):
57.08, 112.00, 120.68,
d
4.92 (s, 4H), 6.80–6.89 (d, 2H, J = 8 Hz), 7.21–7.25 (m, 4H,
underwent a blue shift from 586 nm to 535 nm, indicating that
Zn2+ can displace Cd2+ to form the TPZn-Zn complex (Fig. 1b).
Although displacement assays have been used in cation recogni-
tion [12], ratiometric displacement approaches with two-photon
fluorescent sensor have not been successful. Although there is
some quenching in the presence of Co2+, Ni2+ and Cu2+, the ratio
F535/F586 does not change after addition of other metal ions (Fig. 1b
and c). The dissociation constants (KdTP) for TPZn-Zn calculated
d
122.46, 126.38, 132.04, 137.00, 149.94, 153.11, 157.20, 190.21.
TOF-MS calcd. for C19H17N3[M+H]+: 304.1450, found: 304.1442.
Probe TPZn aldehyde 3 (0.34 g, 1.2 mmol), and NaH (30 mg,
1.5 mmol) were dissolved in 3 mL of THF, and the solution was
cooled to 0 8C under N2. To this solution, Compound 4 (0.428 g,
1.0 mmol) in 9 mL of THF was added dropwise. The reaction
mixture was stirred for 1 h at 0 8C, and then for 12 h at room
temperature, followed by the removal of THF under reduced
pressure. Water was added to the reaction mixture, and the
product was extracted with dichloromethane (4Â 10 mL). The
organic layer was dried with anhydrous Na2SO4 followed by
evaporation of the solvent. The crude product was separated by
column chromatography with a gradient of hexane in dichlor-
omethane (20%–0%) and ethyl acetate in dichloromethane (0%–
50%). The product was obtained as a red powder in 42% yield. 1H
from TP fluorescence titration curves were K11 = 4.86 Â 10À5
,
K12 = 2.01 Â 10À4 (F535) and K11 = 6.47 Â 10À5, K12 = 1.78 Â 10À4 M
(F535/F586), respectively. TPZn shows excellent selectivity for Zn2+
compared with various metal ions and is pH-insensitive in the
biologically relevant pH range, showing no interference in the pH
range 6–8 for TPZn and TPZn-Zn (Fig. 1d).
Physiologically important metal ions which exist in living cells,
such as Ca2+, Mg2+, Na+, and K+, did not give any responses at 50-
fold excess concentration. Most heavy and transition metal ions,
such as Hg2+, Mn2+, and Pb2+, also had no interference. Ni2+, Co2+
and Cu2+ obviously quenched the fluorescence to some extent, a
NMR (400 MHz, CDCl3): d 8.63–8.74 (d, 4H, J = 4.8 Hz), 7.91 (s, 1H),

S. Wu et al. / Chinese Chemical Letters 25 (2014) 93–98
95
300
250
200
150
100
50
a
2+
Zn
b
300
50
250
200
0
150
2+
Cd
100
50
0
2+
2+
Cu
Co
2+
Ni
0
500
550
600
650
700
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
6
4
3
2
1
0
c
d
TPZn-Zn
5
4
3
2
TPZn
0.000
0.001
0.002
0.003
2
4
6
8
10
pH
2+
C [Zn ] (mol/L)
e
6
5
4
3
2
1
0
Zn2+ K+ Na+ Ca2+ Mg2+ Ag+ Cd2+ Mn2+ Ba2+ Pb2+ Fe2+ Cu2+ Co2+ Ni2+ Hg2+
2+
Zn + Metal ions (mol/L)
Fig. 1. (a) Emission spectra with excitation wavelength of 425 nm (isosbestic wavelength) in Tris–HCl (0.01 mol/L, MeCN/water, 50/50, v/v, pH 7.4) in the presence of
increasing concentrations of Zn2+ (0–30 eq.). The concentration of TPZn was 10
mol/L. (b) Fluorescence spectra of TPZn ( ex 425 nm) (10 mol/L) in the presence of different
metal ions (50 mol/L) in Tris–HCl (0.01 mol/L, CH3CN/water, 50/50, v/v, pH 7.4). (c) The saturation titration of TPZn (10
mol/L) with Zn2+, the minimum concentration of
the added Zn2+ is 10 ex 425 nm) to Zn2+ (50
mol/L. (d) The interference of pH in 6–8 for TPZn and TPZn-Zn. (e) Fluorescence responses of TPZn (10 mol/L, mol/L) in the
presence of selected metal ions (50
mol/L) in Tris–HCl (0.01 mol/L, MeCN/water, 50/50, v/v, pH 7.4). From left to right: Zn2+, K+, Na+, Ca2+, Mg2+, Cd2+, Mn2+, Ba2+, Pb2+, Fe2+
Cu2+, Co2+, Ni2+, and Hg2+
m
l
m
m
m
m
m
l
m
m
,
.
problem faced in the other metal ion sensors [13]. The competition
experiments of Zn2+ mixed with the metal ions shown no
existed, enhancement of the ratio of fluorescence intensity (F535
F586) was observed (Fig. 1e).
/
significant variation in the ratio of fluorescence intensity (F535
/
The two-photon action cross sections (dF) of TPZn and TPZn-Zn
were determined by using the two-photon-induced fluorescence
F586). Fortunately, when the heavy quenchers Ni2+, Co2+ and Cu2+

96
S. Wu et al. / Chinese Chemical Letters 25 (2014) 93–98
200
150
100
50
The double-channel fluorescence images at 515–545 nm and 570–
600 nm are shown in Fig. 3. HEK 293 cells incubated with DMEM
containing 5.0 mol/L TPZn for 0.5 h at 37 8C were stained,
indicating that TPZn can permeate into the cells and accumulate in
them. When cells stained with compound TPZn were incubated
DZn
m
DZn-Zn
TSQ-Zn
with Zn(NO3)2 (5.0
mmol/L) in phosphate-buffered saline (PBS) for
0.5 h and washed, the ratio of fluorescence intensities at 515–
545 nm and 570–600 nm increased immediately (Fig. 3) Ratio
imaging. The intensity ratio data were obtained using Image J
(National Institutes of Health) software (Fig. 3). The cells remained
viable and no apparent toxicity and side effects were observed
throughout the imaging experiments (about 2–3 h). These
experiments indicate that TPZn can provide ratiometric detection
for intracellular Zn2+ ions. Therefore, it could be a useful molecular
probe for studying biological processes involving Zn2+ ions within
living cells.
0
700
750
800
850
900
Wavelength (nm)
We further investigated the utility of TPZn in tissue imaging.
TPM images were obtained from a part of an acute rat hippocampal
Fig. 2. TP action spectra of TPZn, TPZN-Zn, and TSQ-Zn.
slice from a 2-day postnatal rat incubated with 10
mmol/L TPZn for
measurement technique [13]. The maximum two-photon action
cross sections of TPZn and TPZn-Zn were 77 GM and 185 GM at
830 nm, respectively (see Fig. 2). Coordination of the Zn2+ to the
ligand that is conjugated to the 1,4-dicyano-2,5-bis(styryl)benzene
core suppressed the ICT process, and this process also contributes
to the fluorescence intensity enhancement, so the two-photon
action cross section of TPZn-Zn is larger than TPZn. The TP action
spectra of the Zn2+ complexes with TPZn in Tris–HCl (0.01 mol/L,
CH3CN/water, 50/50, v/v, pH 7.4) was 46 times larger than those of
TSQ-Zn (Fig. 2). This finding indicates that TPM images for samples
stained with TPZn would be much brighter than those stained with
commercial sensors.
30 min at 37 8C. They reveal intense fluorescence in the CA3
regions as well as the hilus of dentate gyrus (Fig. 4a) [14]. The TPM
images obtained at a depth of 80–160
m ]
m revealed the [Zn2+
distribution in the mossy fibers of dentate granule neurons near
the hilus exclusively in the given plane along the z direction (see
Fig. 5). These findings demonstrate that TPZn is capable of
detecting intracellular free Zn2+ ions at a depth of 80–160
mmol/L
in living tissues by using TPM. Moreover, Fig. 4c–h shows the
results of TPM imaging of the mossy fiber axon terminals of
pyramidal neurons in the CA3 region at a depth of 120
mm. The
double-channel fluorescence images at 515–545 nm and 570–
600 nm both have regions of intense fluorescence, which seemed
to be derived from localization of the dye. However, this influence
was diminished in the ratio image, and higher ratios were clearly
observed in the CA3 region rich in vesicular zinc. Treatment of the
cells with the membrane-permeable metal ion chelator TPEN
We next examined the application of TPZn for ratiometric
fluorescence imaging of Zn2+ in cultured living cells (HEK 293
cells). Two-photon microscopy (TPM) imaging of HEK 293 cells was
observed under a LSM 510 Meta two-photon microscope (Zeiss).
Fig. 3. TPM of live HEK 293 cells. (a and b) Cells incubated with 5.0
(b). (c and d) Cells supplemented with 5.0
mol/L Zn2+ in the growth media for 0.5 h at 37 8C emission collected at 570–600 nm (c) and emission collected at 515–545 nm (d).
The excitation wavelength was 830 nm. TP ratio fluorescence (F535/F586) images of Zn2+ in HEK 293 cells. (Up) HEK 293 cells incubated with compound TPZn (5.0
mol/L).
(Down) HEK 293 cells incubated with compound TPZn (5.0 mol/L) and then further incubate with 5.0 mol/L Zn(NO3)2.
mmol/L TPZn for 0.5 h at 37 8C, emission collected at 570–600 nm (a) and emission collected at 515–545 nm
m
m
m
m

S. Wu et al. / Chinese Chemical Letters 25 (2014) 93–98
97
Fig. 4. TPM images of a part of an acute rat hippocampal slice. (a) Images of living tissue stained with 10
magnification 10Â. (b) Magnification at 100Â shows sin the hilus (H) of dentate gyrus (DG) regions (purple regions of a) at a depth of about 120
emission collected at 515–545 nm using magnification at 100Â in the CA3 regions at a depth of about 120 m before (c) (red regions of a) and after (d) addition of 200
TPEN to the imaging solution. (e and f) Images with emission collected at 570–600 nm using magnification at 100Â in the CA3 regions at a depth of about 120 m before (e)
and after (f) addition of 200 mol/L TPEN to the imaging solution. (g) Ratiometric images generated from (c) and (e). (h) Ratiometric images generated from (d) and (f). The
mmol/L TPZn in ACSF (pH 7) buffer at a depth of ca. 120
m
m with
m
m. (c and d) Images with
mol/L
m
m
m
m
TPEF images were acquired with excitation at 830 nm with a femtosecond pulse.
Fig. 5. TPM images show the hilus (H) of the dentate gyrus (DG) by magnification at 100Â of a rat hippocampal slice stained with 10
mmol/L TPZn at different depths (a:
80
mm, b: 100 mm, c: 120 mm, d: 140 mm, e: 160 mm). The TPEF images were collected at 520–550 nm upon excitation at 790 nm with fs pulses.
reversed the fluorescence ratio enhancements to baseline levels
(Fig. 4h), which confirms that the increase in fluorescence ratio
results from Zn2+ coordination and not from other phenomena,
such as proton flux or sensor photoactivation. These ratiometric
signals disappeared upon addition of TPEN, indicating that TPZn
can detect endogenous Zn2+ in the CA3 region. To the best of our
knowledge, this is the first example of TP ratiometric fluorescence
has higher binding affinity for Zn2+ than for Cd2+ in fluorimetric
experiments. The TP action spectra of TPZn-Zn indicated a dF value
of 185GM at 830 nm, 46 times larger than those of TSQ-Zn. TPZn
was confirmed to be directly applicable to monitor changes in
intracellular Zn2+ in living tissues, owing to its membrane
permeability. TP Ratiometric imaging with TPZn at a depth of
120
slices [9].
m
m could clearly visualize endogenous Zn2+ in hippocampal
imaging at a depth of 120
m
m to visualize endogenous Zn2+ in
hippocampal slices. These experiments indicate that TPZn is an
effective ratiometric probe for biological applications and can
reversibly monitor changes in intracellular Zn2+ in living tissues.
We believe TP ratiometric sensors with these advantages will be a
vital implement in investigations of the biological significance of
Acknowledgments
This work was financially supported by the Introduction
Research Item of Northwest University for Nationalities (No.
xbmuyjrc201110), the Fundamental Research Funds for the
Central Universities (Nos. zyz2012062 and 31920130024), and
the Young and Middle-Aged Scientists Research Fund of Northwest
University for Nationalities (No. 12XB34).
Zn2+
.
4. Conclusion
In this paper, we have developed a new TP ratiometric sensor
TPZn for Zn2+ based on ICT-PET mechanisms, and pH insensitivity
in a large physiological pH range. TPZn is selective against other
biologically competing alkali- and alkaline-earth-metal ions, and
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