A novel ratiometric two-photon fluorescent probe for imaging of Pd2 + ions in living cells and tissues

Article abstract of DOI:10.1016/j.saa.2016.05.013

Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd^{2 +} in biosystems, herein, we reported

Full text of DOI:10.1016/j.saa.2016.05.013

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A novel ratiometric two-photon fluorescent probe for imaging of Pd2+
ions in living cells and tissues
a,b,c,
a
a
c
b
Liyi Zhou
⁎, Shunqin Hu , Haifei Wang , Hongyan Sun , Xiaobing Zhang
a
College of Packaging and Materials Engineering, Hunan University of Technology, Hunan 412007, PR China
Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center
b
for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, PR China
c
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but
also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd in biosystems,
Received 7 March 2016
Received in revised form 5 May 2016
Accepted 11 May 2016
2+
herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-
structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers
by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species
with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of
Available online 12 May 2016
Keywords:
Two-photon
Ratiometric fluorescent probe
Palladium
2
+
Pd ions in living cells and tissues under two-photon excitation (820 nm), which showed large tissue-imaging
depth (19.6–184.6 μm), and a high resolution for ratiometric imaging.
© 2016 Elsevier B.V. All rights reserved.
Fluorescent imaging
Naphthalimide derivative
1
. Introduction
Palladium, one of the platinum-group elements, has adverse effects
expensive instruments and high skilled professionals. In contrast, fluo-
rescent probes can maintain comparable efficiency and accuracy to
avoid these shortcomings. Therefore, they have been widely exploited
by researchers for the detection of a variety of bio-related substances.
To date, only a few fluorescent probes have been reported for quantita-
tively detection of Pd2 concentration [7]. Unfortunately, most of these
fluorescent probes are excited by one-photon excitation with a short
wavelength, which results in some obvious drawbacks for bioimaging,
such as high autofluorescence background, photobleaching, and shal-
low tissue-penetration depth. Moreover, most of these probes are
based on a single emission peak, which tends to be affected by a variety
of factors, including instrumental variations, environmental condition
changes, and probe concentration changes. Therefore, developing a sim-
ple and reliable fluorescent probe for the quantitative detection of the
on our health when it is taken into human bodies via contaminated
food, medicine, and water [1–2]. Therefore, detection and removal of
it have received intense attention for their significance in chemistry, bi-
ology, and environmental science [3]. In particular, palladium, can has
adverse effects on our health and the environment, because DNA, pro-
teins, and other biomacromolecules, such as vitamin B6, have been re-
ported to be able to strongly bind it, which may lead to a variety of
cellular dysfunction processes [4]; additionally, palladium is as well as
often used to prepare dental materials, electric equipment, jewelry,
and automobile exhaust catalysts [5]. Wherefore, this led to the urgent
need to develop an efficient method of detecting Pd2 ions both in
the living and environmental setting.
+
+
2
+
Pd concentration not only in living cells but also in living tissues is
of great significance and necessity.
So far, several analytical techniques have been developed for the ef-
ficient analysis and detection of palladium species, such as inductively
coupled plasma mass spectrometry (ICP-MS), atomic absorption spec-
trometry (AAS), inductively coupled plasma emission spectroscopy
To solve these problems, we used an effective two-photon fluores-
cence microscopy (TPFM) approach, in which a two-photon (TP) fluo-
rescent dye was excited by a long-wavelength laser light and thus
provided a variety of advantages compared with traditional confocal
microscopy [8]. It can improve the three-dimensional imaging of living
tissues, reduce photodamage to biosamples, increase tissue penetration,
and lower background fluorescence. Additionally, associating with a
ratiometric strategy, the built-in correction of the two emission bands
could further eliminate interference caused by instrumental variations,
environmental factors, and probe concentrations [9].
(
ICP-AES), X-ray fluorescence (XRF), and so on [6]. Although these
methods are sensitive towards palladium, they usually require
⁎
Corresponding author at: College of Packaging and Materials Engineering, Hunan
University of Technology, Hunan 412007, PR China.
E-mail address: zhouly0817@163.com (L. Zhou).
http://dx.doi.org/10.1016/j.saa.2016.05.013
1386-1425/© 2016 Elsevier B.V. All rights reserved.

26
L. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
Recently, 4-hydroxy-naphthalimide derivative have been reported
as an efficient two-photon platform for designing two-photon probes
with its tunable two-photon properties because of the hydroxyl group
from 690 nm to 1020 nm with pulse duration of less than 100 fs and a
repetition rate of 80.5 MHz. The laser beam was focused onto the sam-
ples using a lens with a focus length of 3.0 cm. The emission was collect-
ed at an angle of 90° to the direction of the excitation beam to minimize
the scattering. The emission signal was directed into a CCD (Princeton
Instruments, Pixis 400B) coupled monochromator (IsoPlane160) with
an optical fiber. A 750 nm short pass filter was placed before the spec-
trometer to minimize the scattering from the excitation light. The two
photon absorption (TPA) cross section (δ) of the sample (s) at each
wavelength was calculated according to Equation (1), and rhodamine
[
10]. Herein, we used this platform further to develop a new ratiometric
2
+
two-photon fluorescent probe, Np-Pd, for Pd
ion detection and
bioimaging applications. The probe Np-Pd showed two well-separated
fluorescence emission peaks(445 nm and 550 nm), which provided a
large signal-to-background ratio of 26, and therefore high sensitivity
−
7
of the probe with a detection limit of 2.8 × 10
mol/L observed for
2
+
Pd ions. It exhibited a pronounced ratiometric signal changes as the
2
+
deprotection of aryl propargyl ethers in the presence of Pd ions, and
large tissue-imaging depths with a high resolution for ratiometric imag-
ing by TPFM.
3
B in CH OH was used as the reference (r) [12].
δ ¼ δrðSs Φr fr crÞ=ðSr Φs fs csÞ
ð1Þ
2
. Experimental
where S is the integrated fluorescence intensity, Φ is the fluorescence
quantum yield, C is the concentration of sample (s) and reference (r),
and ϕ is the collection efficiency of the experimental setup. The uncer-
tainty in the measurement of cross sections is ~15%. The detailed calcu-
lation is given in the Supplementary data (Fig. S1).
2
.1. Reagents and apparatus
Unless otherwise specified, all chemicals were obtained from com-
mercial suppliers and used without further purification. Thin layer chro-
matography (TLC) was carried out using silica gel 60 F254, and column
chromatography was conducted over silica gel (100–200mesh), both of
them were obtained from Qingdao Ocean Chemicals (Qingdao, China).
In all experiments, water used was doubly distilled and purified by a
Milli-Q system (Millipore, USA). LC-MS analyses were performed
using an Agilent 1100 HPLC/MSD spectrometer. Mass spectra were per-
formed using an LCQ Advantage ion trap mass spectrometer (Thermo
2.4. Spectrophotometric measurements
The fluorescence measurement experiments were measured in
phosphate buffer solution (10 mM) with DMSO as co-solvent solution
(H O/ DMSO = 99:1, v/v). The pH value of PBS solution used was
2
from 3.0 to 10, which was achieved by adding minimal volumes of HCl
solution or NaOH solution. The fluorescent emission spectra were re-
corded at excitation wavelength of 375 nm with emission wavelength
1
Finnigan). HNMR spectra were obtained using a Bruker DRX-400 spec-
−
3
trometer using TMS as an internal standard. All chemical shifts are re-
ported in the standard δ notation of parts per million. UV–vis
absorption spectra were recorded in 1.0 cm path length quartz cuvettes
on a Shimadzu 2450 UV–visible Spectrometer. Fluorescence measure-
ments were carried out on a F4500 fluorescence spectrometer with ex-
citation and emission slits set at 5.0 nm and 5.0 nm, respectively. The pH
was measured with a Mettler-Toledo Delta 320 pH meter.
range from 425 to 650 nm. A 1 × 10
mol/L stock solution of probe
was prepared by dissolving probe compound in DMSO. Procedure of cal-
ibration measurements with probe in the buffer with different pH
followed: 20 μL stock solution of probe and 1980 μL PBS buffer solution
with different pH were combined to afford a test solution, which
−
6
contained 1 × 10 mol/L of probe. The solutions of various testing spe-
cies were prepared from NaCl, CaCl MgSO CuCl ·H O,
Zn(NO ·6H O, using twice-distilled water with final concentrations
2
,
2
,
2
2
)
3 2
2
2
.2. Synthesis of probe molecule Np-O, and Np-Pd
of 0.0125 mol/L, as well as, glutathione (GSH), cysteine (Cys), and gluta-
mate (Glu) using twice-distilled water with final concentrations of
0.025 M. Procedure of selectivity experiments followed: for cations or
anions, 20 μL stock solution of probe, 1948 μL PBS solution (pH 7.4)
and 32 μL solution of each cation or anion were combined to afford a
Synthesis of Np-Br: 2.57 g (0.01 mol) compound 1 and 1.8 g
(
3
0.011 mol) 4-bromo aniline was dissolved in 100 mL of CH COOH,
the mixture was refluxed for 4 h, then poured into ice-water, the solid
was washed with HCl(aq.) and NaOH(aq.), respectively, and then the
gray white solid was obtained via the vacuum pump leak and dried
−
6
test solution, which contained 1 × 10 mol/L of probe and 100 μM cat-
ion or anion; for amino acids, 20 μL stock solution of probe, 1900 μL PBS
buffer solution (pH 7.4) and 80 μL solution of each amino acid were
under the vacuum oven. 1HNMR(400 MHz, d
-DMSO) δ(ppm) =
0.07 (s, 1H), 8.63–8.59 (t, J = 4 Hz, 1H), 8.35–8.26 (t, J = 18 Hz, 1H),
.03 (s, 1H), 7.76–7.74 (d, J = 4 Hz, 1H),7.56–7.54 (d, J = 4 Hz, 1H),
.48–7.39 (m, 4H), +C ESI ms = 432.3, calcd = 431.0.
6
−
6
1
8
7
combined to afford a test solution, which contained 1 × 10 mol/L of
probe and 100 μM amino acid.
According to the literature to synthesize 4-hydroxyl group naphtha-
2.5. HPLC analysis
1
6
lene imide (Np-O) [11]: Np-O: HNMR (400 MHz, d -DMSO) δ(ppm) =
1
1.97 (s, 1H), 8.59–8.53 (3H), 8.71–7.17 (6H), +C ESI ms = 368.4,
calcd = 368.2.
Synthesis of Np-Pd: 184 mg (0.5 mmol) Np-O, 138 mg (1 mmol)
CO , 118 mg (1 mmol) 3-bromoprop-1-yne, and 50 mL CH CN were
added into a round bottom flask, the mixture was reacted for 4 h at
0 °C, then poured into ice-water, the solid was washed with water
1 μM of Np-Pd in DMF (2.0 mL) and 1 μM of Np-O in buffered
(10 mM PBS, pH 7.4) DMF/ H
the control solution. The reaction solution (2.0 mL) was prepared with
5 μM Np-Pd in buffered DMF/H O solution incubated for 30 min at
2
O solution (1:99, v/v) were prepared as
K
2
3
3
2
37 °C after addition of Pd2 ions (50 eq.). An aliquot of each solution
(100 μL) was loaded onto an Inertsil ODS-3 (250 mm × Φ 4.6 mm)
C18 column (GL Sciences, Inc.) fitted on an Agilent 1260 Infinity HPLC
system, and the eluates were monitored with a photodiode array detec-
tor. Detection wavelength was kept at 330 nm, and flow rate was set at
1.0 mL/min. Milli-Q water containing 0.1% TFA (A) and MeCN (B) were
used as developing solvents. Gradient conditions were chosen as fol-
lows: 10% A and 90% B for 15 min.
+
5
three times, and then the white solid was obtained in 95% yield via
the vacuum pump leak and dried under the vacuum oven. 1HNMR
(
400 MHz, d
6
-DMSO) δ(ppm) = 8.59–8.47(m, 3H), 7.89–7.85(t, J =
8
3
Hz, 1H), 7.71–7.67(d, J = 16 Hz, 2H), 7.44–7.35(m, 4H), 5.24(s, 1H),
.33(s, 1H), +C ESI ms = 408.3, calcd = 406.2.
2
.3. Two-photon excited fluorescence measurement
2.6. Cell cytotoxic assays and imaging
The two-photon excited fluorescence was measured by using a Ti:
sapphire femtosecond oscillator (SpectraPhysics Mai Tai) as the excita-
tion source. The output laser pulses have a tunable central wavelength
The cytotoxic effects of the probe were assessed using MTT assays.
Fluorescent images of cells and tissues were obtained using Olympus

L. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
27
FV1000-MPE multiphoton laser scanning confocal microscope (Japan).
For fluorescent imaging, the cells were washed with PBS buffer followed
by incubation with the probe (5 μM) for 30 min in culture medium (PBS
buffer containing 1% DMSO) at 37 °C. Cell samples were then washed
with PBS three times and imaged. Pd2 ions (20 μM) were added to in-
duce a ratiometric signal change during imaging. The two-photon exci-
tation wavelength of the femtosecond laser was fixed at 820 nm; the
emission wavelengths were recorded at (420–460) nm, (520–570)
nm, respectively.
Pd2+ concentration (0–1.0 μM) (Fig. 1D). What's more, in the present
study, the detection limit (3σ/slope) [7h] was estimated to be as low
−
7
2+
as 2.8 × 10 mol/L for Pd , which is sufficiently low for the detection
2
+
of the submillimolar concentration range of Pd found in many chem-
ical and biological systems. The fluorescence enhancement response of
Np-Pd to Pd2 is most likely the result of the conversion of probe Np-
Pd to compound Np-O (Scheme 1). To verify the proposed mechanism,
the change of UV–vis absorption spectra and HPLC spectra of Np-Pd
upon the addition of Pd2 were first investigated (Fig. 1A and Fig. S2).
To further verify this hypothesis, the purified product of the reaction
of Np-Pd with Pd2 ions was then characterized by NMR, which agreed
well with the presynthesized Np-O, directly indicating the correct of our
proposed mechanism.
+
+
+
+
2
.7. Preparation and staining of mouse liver tissue slice
The mouse liver tissue slice were cultured with 10 μM Np-Pd in an
incubator at 37 °C for 1 h and washed with PBS three times, following,
the slices were added 50 μM Pd2 for another 1 h and then washed
with PBS three times for TPFM imaging. The TPFM images (with a mag-
nification at 10×) were collected in two channels (blue = 420–460 nm,
yellow = 520–570 nm) upon excitation at 820 nm with a pulse laser.
+
3.3. Selectivity, Effect of pH and time-dependent fluorescence response
High selectivity and suitable pH working range are an important pa-
rameter to evaluate a newly designed fluorescent probe performance.
Np-Pd was treated with a wide variety of analytes to examine its selec-
tivity. As shown in Fig. 2A (black bars), the addition of Pd2 induced a
significant red-shift of the fluorescence emission spectra. However,
upon treatment with 10 equivalents of other analytes the fluorescence
intensities almost had no changes, even if after a very long reaction
time. So, via the selectivity experiments, we found that only Pd2 ions
could respond to the probe Np-Pd, whereas other ions showed no per-
ceptible effect, demonstrating high selectivity of the probe towards
+
3
. Results and discussion
3
.1. Design and synthesis of probe molecule Np-Pd
+
We employ a previously reported D-π-A-structured 4-hydroxy-
naphthalimide platform to design an efficient ratiometric two-photon
fluorescence probe for Pd2 ions. In our designed probe Np-Pd,
exhibiting a pronounced ratiometric signal changes as the deprotection
of aryl propargyl ethers in the presence of Pd2 ions (Scheme 1). On the
basis of previously reported, Np-Pd was prepared in 95% yield by the
coupling of Np-O with 3-bromo-1-propyne in a one-pot method. The
detailed synthetic procedure is described in Scheme 1, and all the chem-
+
2
+
Pd
ions. Competition experiments were also carried out to assess
+
the practical applicability of the probe. Ten equivalent of other metal
2
+
ions or neutral molecules is added to 50 μM of Pd
separately, and
the fluorescence response of the probe is then recorded with the results
shown in Fig. 2A (red bars). The probe showed almost unchanged fluo-
1
2+
ical structures of compounds were verified by Mass, H NMR spectros-
rescent responses to Pd before and after the addition of other inter-
copy, seen in the Supporting information.
fering metal ions or neutral molecules. These results demonstrated
that our Np-Pd probe could meet the selective requirements for practi-
cal applications.
3
.2. Optical property and selectivity of Np-Pd
We also studied the effect of pH on Np-Pd in the absence and pres-
ence of Pd2 ions (Fig. 2B). Without Pd ions, no obvious characteris-
+
2+
The spectroscopic properties of Np-Pd (5 μM) were examined in
phosphate buffer solution (10 mM, pH 7.4, 99:1 H
different Pd ion concentrations. The probe displays sensitive absorp-
2
O/DMSO, v/v) with
tic fluorescence of the acceptor could be observed from pH 3.0 to 10.0.
2
+
2+
2+
Upon addition of Pd ions, the best response towards Pd could be
achieved with a pH range from 4.0 to 10.0. Thus, the PBS solution
(pH 7.4) was used throughout the experiment. These results indicated
that the probe was favorable for applications in practical samples at dif-
ferent pH values. What's more, Np-Pd possesses a quick fluorescent re-
sponse time within 5 min, which could ensure detection of low
concentration of Pd2 in living systems with a fast response time (Fig.
2C).
2
+
tion (Fig. 1A) and fluorescence (Fig. 1B) responses to changes in Pd
ion concentration. As shown in Fig. 1A, in the absence of Pd2 ions,
only one absorption peak was observed at the maximum absorption
wavelength (λ = 350 nm). After Pd2 ions (0–50 μM) were added, a
new absorption peak appeared at 470 nm. Further fluorescence experi-
+
+
+
ments showed a dramatic increase in fluorescence intensity at λem
=
=
5
4
50 nm and an obvious decrease in fluorescence intensity at λem
45 nm (Fig. 1B). F550/445 was gradually increased from 0.16 to 4.2
2
+
with the increasing concentration of Pd changed from 0 to 50 μM, re-
spectively, corresponding to a signal-to-background ratio of 26, which
provided the basis for high-performance ratiometric (F550/F445) detec-
tion and exhibited an excellent F550/F445 linearity towards different
3.4. Two-photon active absorption cross-section measurement
Naphthalimide derivative exhibit excellent two-photon properties
with a two-photon action cross-section, showing this two-photon dye
Scheme 1. Structure of Np-Pd and its response mechanism to palladium species.

28
L. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
2
+
2+
Fig. 1. (A) Absorption spectra of 5 μM Np-Pd and 5 μM Np-Pd + 50 μM Pd ions; (B) Fluorescence spectra of 5 μM Np-Pd in the presence of increasing concentrations of Pd ions (0–
2
+
2+
5
0 μM); (C) Calibration curve of Np-Pd to Pd ions. The curve was plotted with the fluorescence intensity vs Pd ion concentration (0–50 μM); (D) Linear relationship between F550/F445
and Pd ion concentration in the 0–1.0 μM range; (E and F) Change in color of the probe before and after the addition of 50 μM Pd ions in 5 μM of Np-Pd in 99:1 PBS/DMSO, pH 7.4; (G
and H) Change in the fluorescence of the probe before and after the addition of 50 μM Pd ions in 5 μM of Np-Pd under excitation by UV light.
2
+
2+
2
+
2
+
Fig. 2. (A) Fluorescence response of 5 μM Np-Pd to 50 μM of Pd or 10 equivalents of other metal ions or neutral molecules (pink bars) and to the mixture of 10equivalents of other
2
+
2+
divalent metal ions or neutral molecules with 50 μM of Pd (brick red bars); (B) pH effects on Np-Pd in the absence or presence of Pd . The numbers from 1 to 16 correspond to
GSH to Fe3+, respectively(GSH, Cys, Glu, Pb , Hg , Ca2+, Mg2+, Co , Cu , Pd , Ni , Cr , Zn , Al , Na , and Fe ; (C) Time-dependent fluorescence response records of
μM Np-Pd in 10 mM PBS solution (1% DMSO, pH 7.4) upon additions of 50 μM Pd ions solution. (For interpretation of the references to color in this figure legend, the reader is
2+
2+
2+
2+
2+
2+
3+
2+
3+
+
3+
2
+
5
referred to the web version of this article.)

L. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
29
2
+
Fig. 3. TP-excited ratiometric fluorescence images of 5 μM Np-Pd in 10 mM PBS in HeLa cells stimulated with or without Pd ions. TP images from the blue channel (A) and the yellow
2
+
channel (B) without Pd ions; (C) Merged images of (A) and (B); (D) Ratiometric images of (A) and (B); (E) Two-photon images from the blue channel and (F) the yellow channel with
2
+
Pd ions; (G) Merged images of (E) and (F); (H) Ratiometric images of (E) and (F). Two-photon images: λex = 820 nm. Blue channel: λem = 420–460 nm; yellow channel: 520–570 nm.
Scale bar: (A–D) 50 μm, and (E–H) 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
is potentially useful for bioimaging applications. For our probe Np-Pd, in
the presence of Pd2+ ions, the Np-O was calculated to has a two-photon
active absorption cross-section of 70GM, as well as a new strong fluo-
rescent peak appeared at 550 nm (Fig. S1, the black line).
2
+
the absence of Pd ions, the Np-Pd was calculated to have a two-pho-
ton active absorption cross-section of 78GM (1GM = 10− (cm s)/
50
4
photon) at 445 nm upon excitation at 820 nm (Fig. S1, the red line). In
Fig. 4. TPFM images of a frozen liver tissue slice from a nude mouse stained with 10 μM Np-Pd at 69.6 μm for 60 min followed by treatment with 50 μM Pd2+ and incubated for another
6
0 min (A, B); (C) Overlay image of (A) and (B); (D) Ratiometric image of (A) and (B). The images were collected at 420−460 nm (blue channel, A) and 520−570 nm (yellow channel, B)
upon excitation at 820 nm with femtosecond pulses. Scale bar: 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

30
L. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 166 (2016) 25–30
3
.5. Tatiometric two-photon imaging in living cells
To demonstrate the practical applicability of the probe in biological
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.saa.2016.05.013.
systems, we carried out fluorescence imaging experiments in living
cells (HeLa cells). The cytotoxicity of the probe was first evaluated by
using MTT assays for HeLa cells. Experimental results demonstrated
that the Np-Pd probe exhibited negligible cytotoxicity to the cell lines
References
(
Fig. S3 in the Supporting information). Before two-photon fluorescent
[
1] (a) J.C. Wataha, C.T. Hanks, J. Oral Rehabil. 23 (1996) 309–320;
b) J. Kielhorn, C. Melber, D. Keller, I. Mangelsdorf, Health 205 (2002) 417–432.
[2] B. Liu, H. Wang, T. Wang, Y. Bao, F. Du, J. Tian, Q. Li, R. Bai, Chem. Commun. 48 (2012)
867–2869.
imaging, cells were incubated with 5 μM Np-Pd at 37 °C for 30 min,
which then exhibited strong two-photon intracellular fluorescence in
blue channel (Fig. 3A) and weak fluorescence in the yellow channel
(
2
[
3] T. Schwarze, H. Müller, C. Dosche, T. Klamroth, W. Mickler, A. Kelling, H.G.
Löhmannsröben, P. Saalfrank, H.J. Holdt, Angew. Chem. Int. Ed. 46 (2007)
1671–1674.
(
Fig. 3B). These results demonstrated that Np-Pd could penetrate the
2
+
cell membrane. However, incubation of the cells with 20 μM of Pd
[
4] (a) T. Gebel, H. Lantzsch, K. Plebow, H. Dunkelberg, Mutat. Res. Genet. Toxicol. En-
viron. Mutagen. 389 (1997) 183–190;
ions could significantly decrease the two-photon excited fluorescence
signal in the blue channel (Fig. 3E) but with an enhancement of the
two-photon excited fluorescence signal in the yellow channel (Fig.
(b) C.L. Wise-man, F. Zereini, Sci. Total Environ. 407 (2009) 2493–2500;
(c) C.D. Spicer, T. Triemer, B.G. Davis, J. Am. Chem. Soc. 134 (2012) 800–803;
(d) R.M. Yusop, A. Unciti-Broceta, E.M.V. Johansson, R.M. Sánchez-Martín, M.
Bradley, Nat. Chem. 3 (2011) 239–243.
3
F). These preliminary experimental results demonstrated that Np-Pd
could be successfully applied for two-photon-excited ratiometric imag-
ing of Pd ions in live cells.
[
[
5] M. Paraskevas, F. Tsopelas, M. Ochsenkühn-Petropoulou, Microchim. Acta. 176
2
+
(
2012) 235–242.
6] (a) O.V. Borisov, D.M. Coleman, K.A. Oudsema, R.O. Carter III, J. Anal. At. Spectrom.
2 (1997) 239–246;
b) B. Dimitrova, K. Benkhedda, E. Ivanova, F. Adams, J. Anal. At. Spectrom. 19
2004) 1394–1396;
1
3
.6. Tissues slice ratiometric imaging
(
(
Np-Pd was further applied for TP-excited fluorescence imaging of
(c) C. Locatelli, D. Melucci, G. Torsi., Anal. Bioanal. Chem. 382 (2005) 1567–1573;
(d) K. Van Meel, A. Smekens, M. Behets, P. Kazandjian, R. VanGrieken, Anal. Chem.
Pd2+ ions in liver tissue slices from nude mice with images at different
79 (2007) 6383–6389.
tissue depths recorded by TPFM in the Z-scan mode. Experimental re-
sults indicated that the probe could be successfully applied for
[
7] (a) S. Sun, B. Qiao, N. Jiang, J. Wang, S. Zhang, X. Peng, Org. Lett. 16 (2014)
1132–1135;
2
+
(b) F. Song, A.L. Garner, K. Koide., J. Am. Chem. Soc. 129 (2007) 12354–12355;
(c) A.L. Garner, F. Song, K. Koide, J. Am. Chem. Soc. 131 (2009) 5163–5171;
(d) W. Guo, L. Wang, J. Jiao, J. Hou, Y. Cheng, C. Zhu, Tetrahedron Lett. 53 (2012)
3459–3462;
ratiometric imaging of Pd ions in tissue at a depth of 19.6–184.6 μm
in dul-channels at two well-separated wavelengths (see Fig. 4 and Fig.
S4 in the Supporting information). These results demonstrated that
the probe possessed a good staining capability and high penetrating
ability in tissue as well as high resolution for two-color ratiometric
imaging.
(e) M. Yang, Y. Bai, W. Meng, Z. Cheng, N. Su, B. Yang, Inorg. Chem. Commun. 46
2014) 310–314;
(
(f) J. Zhang, L. Zhang, Y. Zhou, T. Ma, J. Niu, Microchim. Acta 180 (2013) 211–217;
(g) Y. Zhou, J. Zhang, H. Zhou, Q. Zhang, T. Ma, J. Niu., Sensors Actuators B Chem.
171-172 (2012) 508–514;
(
(
h) B.C. Zhu, C.C. Gao, Y.Z. Zhao, C.Y. Liu, Y.M. Li, Q. Wei, Z.M. Ma, B. Du, X.L. Zhang,
Chem. Commun. 47 (2011) 8656–8658;
i) H. Li, J. Fan, X. Peng, Chem. Soc. Rev. 42 (2013) 7943–7962;
4
. Conclusion
In summary, we have designed and synthesized a novel robust
(j) H. Cui, H. Chen, Y. Pan, W. Lin, Sensors Actuators B 219 (2015) 232–237;
(k) W. Liu, J. Jiang, C. Chen, X. Tang, J. Shi, P. Zhang, K. Zhang, Z. Li, W. Dou, L. Yang,
ratiometric two-photon fluorescent probe, termed Np-Pd, for detecting
W. Liu, Inorg. Chem. 53 (2014) 12590–12594.
2
+
Pd ions not only in living cells but also in living tissues. Np-Pd is based
on a two-photon fluorophore 4-hydroxyl group naphthalimide. In
probe Np-Pd, it shows a ratiometric signal changes as the deprotection
of aryl propargyl ethers in the presence of Pd2 ions. The experiments
demonstrate that Np-Pd possesses high ratiometric imaging resolution
and large tissue-imaging depth (19.6–184.6 μm) at the cellular and tis-
sue levels. We believe that Np-Pd could find wide applications not only
in environmental monitoring but also biomedical diagnostics for Pd2
ions.
[
8] (a) H.M. Kim, B.R. Cho, Chem. Asian. J. 6 (2011) 58;
(b) H.M. Kim, B.R. Cho, Acc. Chem. Res. 42 (2009) 863–872.
9] (a) Z.C. Xu, K.H. Baek, H.N. Kim, J.N. Cui, X.H. Qian, D.R. Spring, I.J. Shin, J.Y. Yoon, J.
Am. Chem. Soc. 132 (2010) 601–610;
[
+
(
(
(
b) Z.X. Han, X.B. Zhang, Z. Li, Y.J. Gong, X.Y. Wu, Z. Jin, C.M. He, L.X. Jian, J. Zhang, G.L.
Shen, R.Q. Yu, Anal. Chem. 82 (2010) 3108–3113;
c) Y. Zhao, X.B. Zhang, Z.X. Han, L. Qiao, C.Y. Li, L.X. Jian, G.L. Shen, R.Q. Yu, Anal.
Chem. 81 (2009) 7022–7030;
d) C.Y. Li, X.B. Zhang, L. Qiao, Y. Zhao, C.M. He, S.Y. Huan, L.M. Lu, L.X. Jian, G.L. Shen,
R.Q. Yu., Anal. Chem. 81 (2009) 9993–10001.
+
[10] (a) T. Liu, X. Zhang, Q. Qiao, C. Zou, L. Feng, J. Cui, Z. Xu., Dyes Pigments 99 (2013)
37–542;
5
(
b) Z.R. Dai, G.B. Ge, L. Feng, J. Ning, L.H. Hu, Q. Jin, D.D. Wang, X. Lv, T.Y. Dou, J.N. Cui,
Acknowledgements
L. Yang., J. Am. Chem. Soc. 137 (2015) 14488–14495.
[
[
11] W. Shu, L. Yan, J. Liu, Z. Wang, S. Zhang, C. Tang, C. Liu, B.D. Zhu, Ind. Eng. Chem. Res.
4 (2015) 8056–8062.
12] (a) C. Xu, W.W. Webb, JOSA. B 13 (1996) 481–491;
b) M.A. Albota, C. Xu, W.W. Webb, Appl. Opt. 37 (1998) 7352–7356.
5
This work was supported by Natural Science Foundation of China
NSFC, Grants 21202042), Hunan Provincial Natural Science Foundation
(
(
of China (Grant 13JJ4090).