A selective and sensitive off–on probe for palladium and its application for living cell imaging

Article abstract of DOI:10.1016/j.cclet.2015.09.003

A new rhodamine B derivative T1 has been rationally synthesized and displayed selective Pd(II)-amplified absorbance and fluorescence emission above 540 nm in methanol–water. Upon the addition of Pd(II), the spirolactam ring was unfolded and a 1:1 metal-ligand complex formed, which can be used for “naked-eyes” detection. In addition, fluorescence imaging experiments of Pd²⁺ in HepG2 living cells showed its valuable application in biological systems.

Full text of DOI:10.1016/j.cclet.2015.09.003

Accepted Manuscript
Title: A selective and sensitive off-on probe for palladium and
its application for living cell imaging
Author: Ying Long Jia Zhou Mei-Pan Yang Bing-Qin Yang
PII:
S1001-8417(15)00354-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2015.09.003
CCLET 3426
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Chinese Chemical Letters
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31-3-2015
10-6-2015
15-6-2015
Please cite this article as: Y. Long, J. Zhou, M.-P. Yang, B.-Q. Yang, A selective and
sensitive off-on probe for palladium and its application for living cell imaging, Chinese
Chemical Letters (2015), http://dx.doi.org/10.1016/j.cclet.2015.09.003
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Original article
A selective and sensitive off-on probe for palladium and its application for living cell imaging
Ying Long, Jia Zhou, Mei-Pan Yang, Bing-Qin Yang yangbq@nwu.edu.cn
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, College
of Chemistry and Materials Science, Northwest University, Xi’an 710069, China
Corresponding author.
Abstract
A new rhodamine B derivative T1 has been rationally synthesized and displayed selective Pd(II)-amplified absorbance and fluorescence emission
above 540 nm in methanol-water. Upon the addition of Pd(II), the spirolactam ring was unfolded and a 1:1 metal-ligand complex formed, which can be
used for “naked-eyes” detection. In addition, fluorescence imaging experiments of Pd2+ in HepG2 living cells showed its valuable application in
biological systems.
Keywords
Rhodamine B, Palladium, Fluorescence probe, Living cell imaging
1. Introduction
Fluorescence probe has become a widely used and important tool for monitoring metal ion concentration in biological samples. Most
sensors reported have a metal chelating site linked to a fluorophore, and the metal binding causes a change in fluorescence intensity [1-
6]. The development of synthetic receptors enabled the recognition of important metal ions, especially transition-metal ions, in
biologically and environmentally relevant samples. This has attracted widespread interest from biologists, chemists, environmentalists
and clinical biochemists in recent years [7–12]. The metal ions, like platinum, mercury, zinc and palladium have environmental and
biological importance.
Palladium belongs to the platinum-group elements (PGEs; consist of Pt, Pd, Ru, Rh, Ir and Os,). It is widely used in various materials
such as catalysts, dental crowns, jewelry and fuel cells [13-15]. Pd-catalyzed reactions such as the Heck, Sonogashira, Buchwald–
Hartwig and Suzuki–Miyaura reactions represent powerful transformations for the synthesis of complex molecules, which played an
important role in pharmaceuticals [16-23]. However, their fruitful and frequent use can also result in the contamination of soil and
water systems [23] and therefore could cause health hazards [24-26]. The restrictions of government on the residual heavy metals in
products are very critical. Therefore, palladium detection both in living systems and in environment has attracted tremendous attention.
Detecting the content of palladium was usually carried out by atomic absorption/emission spectroscopy, ion-coupled plasma emission-
mass spectroscopy, solid-phase microextraction/high performance liquid chromatography, and X-ray fluorescence spectroscopy [27-
28]. Although these conventional methods provide an extremely sensitive and rapid analysis, they need sophisticated sample-
pretreatment procedures, complicated instrumentation and rigorous experimental conditions.
In recent years, the fluorescence method has been developed for palladium analysis and the colorimetric technique has
frequently been applied [29-31]. For example, palladium could be detected by fluorescent ligands via fluorescence quenching [32-38].
Holdt et al. designed the first chemical sensors for Pd2+ detection through increasing fluorescence [39].
Rhodamines are dyes widely employed in the study of complicated biological systems as fluorescence probes due to their high
fluorescence quantum yields, high absorption coefficients, long-wavelength emissions and absorptions [40]. Owing to the spirolactam
scaffold in rhodamines, which undergoes a conformational transformation from the spirolactam (nonfluorescent and colorless) to an
open-ring structure (fluorescent and colored), they have been widely applied in the design of chemical sensors in recent years, and
these new designed chemosensors have been reviewed recently [41-44]. In this spirocyclic form, the sensing event occurs in its five-
membered lactam moiety. Obviously, these properties still present abundant opportunities for the design of new fluorescent probes.
Our research work encompasses the design, synthesis, spectroscopy and biological applications of fluorescent chemosensors for
selective sensing of Pd2+. Here we are reporting a novel rhodamine based chemosensor T1, which was prepared by a three-step
synthesis using inexpensive materials. Particularly, the chemosensor showed highly selective and sensitive fluorescence “turn-on”
behaviors toward Pd2+ followed the remarkable color changes from colorless to pink, which can be used for “naked eyes” detection.
Moreover, the probe can give highly selective spectroscopic responses to Pd2+ over other metal ions in living cells, which showed T1
can penetrate cell membranes and react with Pd2+ within living cells.
Page 1 of 7

2. Experimental
2.1 Materials
All the solvents and reagents were of analytic grade and they were used without further purification unless for special needs. The T1
was dissolved in DMF-MeOH in concentration of 1mmol/L as stock solution. And then quantificational T1 was used for different
testing systems. We used the HITACHI F-4500 fluorescence spectrophotometer to measure the fluorescence spectra. And the
absorbance spectra measurements were also performed on a Shimadzu UV-1700 spectrophotometer. IR spectra were collected on a
Bruker Tensor 27 spectrometer. NMR spectra were recorded on a Varian INOVA -400 MHz spectrometer (at 100 MHz for 13C NMR
and 400 MHz for 1H NMR). A Bruker micro TOF-Q II ESI-TOF LC/MS/MS Spectroscopy was used to perform mass spectra. And the
living cells imaging was performed on an Olympus FV1000 confocal microscope. We set the excitation wavelength as 540 nm. All
reagents used for synthesis were of analytical-reagent grade and commercially available from Aldrich. Thin-layer chromatography
(TLC) and column flash chromatography were performed using silica gel GF254 and Merck silica gel(250–400 mesh ASTM),
respectively. And the twice-distilled water was used throughout the experiment.
2.2 Synthesis
Synthesis of compound 2: We synthesized the compound 2 from Rhodamine B using the procedures published in literature [45-48]
.
Synthesis of compound 3: In a 100 mL flask, an excess of isophthalaldehyde (0.268 g, 0.002 mol) was dissolved in 20 mL of methanol,
cooled in a cold water bath. Then Rhodamine hydrazide (0.461g, 0.001mol) was dissolved in 30 mL of methanol and added dropwise
to the above solution with vigorous stirring, and the stirred mixture was allowed to stand in the cold water bath for about 4h, then the
solvent was removed under reduced pressure, the crude was purified by silica gel column chromatography to give 3 (white powder) in
78.8% yield. MALDI-TOF MS calcd. for (C36H36N4O3) m/z = 573.2860( M++1), Found: 573.2802. 1H NMR (400 MHz, CDCl3) δ: 1.13
(t, 12H, J = 8 Hz), 3.24~3.29 (m, 8H), 6.41~6.43 (m, 2H), 6.63~6.67 (m, 4H), 7.11 (d, 1H, J = 8 Hz), 7.37 (t, 1H, J = 8 Hz), 7.53~7.57
(m, 3H), 7.73 (d, 1H, J = 8 Hz), 7.98 (d, 1H, J = 8 Hz), 8.16 (d, 1H, J = 8 Hz), 9.18 (s, 1H), 9.64 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ: 192.2, 165.2, 153.1, 151.9, 149.0, 144.8, 136.6, 136.4, 133.6, 132.8, 129.9, 129.4, 129.0, 128.9, 128.4, 127.9, 123.9, 123.5, 108.0,
105.8, 97.9, 44.3, 12.6.
Synthesis of Compound 4: Compound 3 (0.572 g, 0.001 mol) was dissolved in 20 mL of methanol. Then a solution of 2-aminophenol
(1.2g, a little excess) in methanol (20 mL) was added and the mixture was stirred for 2 h at 60 oC. Then the precipitates were collected
and washed 3 times with 10 mL of cold ethanol. After drying under reduced pressure, The crude product was purified by
recrystallization in CH3CN/H2O to give compound 4 (white solid) in 82.6% yield. IR (KBr/cm-1) υ: 3444, 2967, 2925, 1694, 1615,
1550, 1512, 1460, 1426, 1356, 1307, 1270, 1223, 1120, 1072, 1011, 980, 946, 873, 820, 786, 755, 688 cm-1. MALDI-TOF MS calcd.
for (C42H41N5O3) m/z = 663.3282, Found: 663.3298. 1H NMR (400 MHz, CDCl3) δ : 1.15 (t, 12H, J = 8 Hz), 3.30~3.35 (m, 8H),
6.23~6.27 (m, 2H),6.49~6.54 (m, 4H), 6.92 (t, 1H, J = 4 Hz), 7.02 (d, 1H, J = 4 Hz), 7.12~7.17 (m, 2H), 7.28~7.31 (m, 1H), 7.37~7.49
(m, 3H), 7.77~7.82 (m, 2H), 7.97~8.03 (m, 2H), 8.64 (s, 1H), 8.82 (s, 1H), 9.96 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 12.6, 44.1,
66.2, 97.9, 105.8, 115.1, 120.1, 123.9, 127.9,129.0, 135.9, 145.9, 151.5, 152.4, 165.0.
2.3 Preparation of the test solution
The 10 µmol/L stock solution of probe T1 was prepared in methanol and water (4/6, v/v). The solutions of various testing cation
species were prepared from CaCl2, AgNO3, MgCl2, CoCl2·6H2O, CdCl2, ZnCl2, CuCl2·2H2O, MnSO4·H2O, HgCl2, NiCl2·6H2O,
Pb(NO3)2, CrCl3·6H2O,PdCl2,BaCl2·2H2O, NaCl, AlCl3·6H2O and FeCl3·6H2O in the doubly distilled water. Before spectroscopic
measurements, the corresponding solutions of 4 were freshly prepared by diluting the high concentration stock solution. All the
measurements were made according to the procedures as follows. Placing 1mL of the probe solution and an appropriate aliquot of each
metal stock into a 10 mL glass tube, and diluting the solution to 10mL with methanol-water (4/6, v/v). The absorbance was at 550 nm
and the fluorescence emission appeared at 583nm. Both the excitation and emission wavelength band passes were set as 5.0nm and the
excitation wavelength was set at 540 nm.
2.4 Cell culture and fluorescence imaging
The human cancer cell line HepG2 (liver cells) were cultured in RPMI 1640 replenished with 10% FBS. Before the experiments, cells
were preprocessed with probe T1 (10 µmol/L) for 1 h at 37 oC in humidified air and 5% CO2, washed three times with PBS then
imaged. After incubation with Pd2+ (10 µmol/L) for another 1.5 h at 37 oC, cells were washed 3 times with PBS to remove remaining
Pd2+ and then imaged. Confocal fluorescence imaging was carried out using an Olympus FluoView FV1000 laser scanning microscope
with a 40× objective lens. Excitation of 1-loaded cells at 540nm was performed with a solid laser and emission was collected at 550–
750 nm.
3. Results and discussion
3.1 Spectroscopic properties
It is an essential feature for a probe to have a high selectivity toward the analyte over the potentially competing species. Shift of
fluorescence and absorbance spectra caused competition and selectivity by T1 in the presence of Pd2+ and other competitive metal ions
were recorded in Figs.1a and b, respectively. No obvious fluorescence changes were observed in the presence of other cations (Fig.1 c),
such as heavy and transition metal ions (Mn2+, Pb2+, Cd2+, Co2+, Fe3+, Cu2+, Ni2+, Ag+, Zn2+, Cr3+, Hg2+), alkali or alkaline-earth metals
(Na+, Ca2+,Mg2+, Ba2+) and Al3+.
Page 2 of 7

However, the F/F0 value increased by more than 100-fold after the addition of Pd2+ at the same concentration. Moreover, Pd2+ caused
remarkable enhancement in fluorescence and obvious color changes. The colorimetric and fluorometric behaviors of T1+Pd2+ complex
could be conveniently distinguished from those of the free T1 by a naked eye, as shown in Fig.2. This strongly suggested that T1 has
high selectivity to bivalent palladium ion and can serve as a “naked- eye” probe for Pd2+.
A fluorescence and UV titration experiment was carried out to further investigate the interactions between T1 and Pd2+. The stable
“spirolactam form” of rhodamine, free T1 represents colorless and no fluorescence response in the range from 550 to 750 nm.
However, upon the gradual addition of Pd2+, a significant enhancement of fluorescence with an emission maximum at 583nm (Fig.3a)
and an absorbance maximum at 550nm (Fig.3.b) was observed. It was attributable to the delocalization effects in the xanthene moiety
of the rhodamine. With the Pd2+ concentration increasing, a continuous increase of fluorescence intensity appeared. After the addition
of 1.0 equiv. of Pd2+, the fluorescence intensity showed negligible changes (Fig. 3a, inset). The results clearly indicated that, owing to
the addition of Pd(II) to T1 ,the spirolactam opened and formed a highly delocalized π-conjugated structure. Thus, an obvious and
significant enhancement of absorbance and fluorescence was observed.
The association constant K was calculated according to the Benesi–Hildebrand equation [49]: (Fmax − F0)/(Fx − F0) = 1 + (1/K) (1/[T]n),
where Fmax, F0, and Fx are fluorescence intensities of the probe in the presence of Pd2+ at saturation, free probe, and any intermediate
Pd2+ concentration (Fig. 4). The binding constant value was found to be K= 8.77 × 104 (mol/L)-1. The linearity of this plot within the
range of 1 µmol/L to 5 µmol/L reveals the formation of a 1:1 clathrate between T1 and Pd2+ (R2=0.9954). When the fluorescence
enhancement reached the maximum of the probe T1 to Pd2+, F/F0 was as high as over 100-fold, demonstrating that T1 has the excellent
capability of detecting Pd2+ both quantitatively and qualitatively.
It is well known that the spirolactam ring of the rhodamine derivatives can open in acidic media and then exhibits the strong
fluorescence of rhodamine. Therefore, it is necessary to evaluate the effect of pH on the fluorescence of T1. The effects of pH were
evaluated in the pH range from 2.0 to 12.0 (Fig. 5). No obvious enhancement of fluorescence at 550 nm was observed within pH 5.0–
12.0, suggesting that it was insusceptible to the change of acid–base solution. However, in the presence of Pd2+, a remarkable
fluorescence emission band at 583 nm formed under different pH values. It showed the pH values of approximately 4~8 gave the
highest responses, suggesting that the probe T1 for Pd2+ could work well in under physiological conditions with a low background
response. Therefore, further studies were carried out in a methanol aqueous solution (4/6, v/v) at pH 7.0. Furthermore, the time-
dependence of probe T1 fluorescence was also evaluated in the presence of Pd2+ ions. The results showed that, upon the reaction of
probe T1 with Pd2+, the fluorescence of all the tested solutions remarkably increased to their maximum value within the first 1 min.
(Fig. 5 insert.).
3.2 Reversibility and mechanism for the binding of T1 with Pd2+
As is well known, the reversibility is an important property for an excellent probe. Thus, the EDTA–adding experiments were
conducted to examine the reversibility of the probe T1. As clearly shown in Fig.6a, the absorbance decreased when EDTA was added
to the mixture containing T1 and Pd2+. Besides, the color of mixture changed from black to colorless and the fluorescence intensity
decreased. When Pd2+ was added to the system again, the signals were almost completely reproduced (Fig. 6b) and the colorless
solution turned to pink. The above process can be repeated several cycles without significant changes in the fluorescence spectrum (Fig.
6b insert). These findings indicated that probe T1 is a reversible fluorescent probe toward Pd2+
.
Binding analysis using the method of continuous variations (Job’s plot) was measured. A maximum absorbance at 550nm(Fig.7 insert)
and fluorescence emission at 583nm(Fig.7) were observed when the molecular fraction of T1 was close to 0.5, which established a 1:1
stoichiometry between T1 and Pd2+. Thus, the most likely binding sites for Pd2+ are the conjugated moieties including phenolic
hydroxyl O, imino N, benzoyl hydrazine N and O atoms (Scheme 2). The chelation–induced ring opening of rhodamine spirolactam
rather than other possible reactions was likely the mechanism [42, 50].
To further demonstrate the practical applicability of the probe in biological samples, fluorescence imaging experiments were conducted
in living cells. The fluorescence images of HepG2 cells were recorded before and after addition of Pd2+, shown in Fig. 8. In the absence
of Pd2+, free T1 showed no detectable fluorescence signals in living cells (Fig. 8a). By contrast, after incubation with Pd2+, a bright
fluorescence was observed in living cells (Fig. 8c). Bright-field transmission images of cells treated with Pd2+ and probe revealed that
the cells were viable throughout the imaging experiments (Fig. 8b). The results suggested that probe T1 can penetrate the cell
membrane and can be applied for in vitro imaging of Pd2+ in living cells and potentially in vivo.
4. Conclusion
Page 3 of 7

In summary, we have described a new rhodamine-based probe T1, which can give reversible, selective, and sensitive fluorescence
enhancement response to Pd2+ via a 1:1 binding mode in methanol aqueous solutions. High selectivity toward Pd2+ is exhibited and
little cross-sensitivity is observed to other commonly coexistent metal ions.
The molecular design might greatly contribute to the development of more efficient and useful probes based on the rhodamine scaffold.
The value of probes in biological systems is demonstrated by the fluorescence imaging in HepG2 cells. It is anticipated that the probes
may be widely used in the studies on the effects of Pd2+ in biological systems.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21172178)
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Scheme 1 Synthesis of 4
Scheme 2 The most likely binding sites for Pd2+
Fig.1 Fluorescence (a) and absorbance (b) spectra of T1 (10 µmol/L) in methanol-water (4/6, v/v) solution upon addition of various metal ions (10 µmol/L),
λex=540 nm. (c) Fluorescence intensity changes of T1 (10 µmol/L) upon the addition of various metal ions (10 µmol/L) in the presence of Pd2+ (10 µmol/L) in
methanol-water (4/6, v/v) solution. The black bars represent the fluorescence response of T1 and competing ions: 1.Al3+，2.Na+, 3.Ca2+, 4.Mg2+, 5.Cd2+, 6.Mn2+
,
7.Ni2+, 8.Ba2+, 9.Zn2+, 10.Co2+, 11.Cr3+, 12.Pb2+, 13.Ag+, 14.Fe3+, 15.Hg2+, 16.Cu2+, 17.Pd2+. The red bars represent the subsequent addition of 10 µmol/L Pd2+ to
the above solutions.
Fig.2 (a) The color change of the probe T1 in different metal ions (1.Al3+，2.Na+, 3.Ca2+, 4.Mg2+, 5.Cd2+, 6.Mn2+, 7.Ni2+, 8.Ba2+, 9.Zn2+, 10.Co2+, 11.Cr3+,
12.Pb2+, 13.Ag+, 14.Fe3+, 15.Hg2+, 16.Cu2+, 17.Pd2+); (b)The fluorescence change of the probe T1 in different metal ions(both 10 µmol/L)
Fig.3 (a) Fluorescence intensity changes of T1 (10 µmol/L) upon addition of Pd2+ in methanol-water (4/6, v/v) solution. Inset: Changes in the emission intensity
at 583nm. (b) Absorbance intensity changers of T1 (10 µmol/L) upon addition of Pd2+ in methanol-water (4/6, v/v) solution.
Fig.4 Determination of binding constant of T1(10 µmol/L) with Pd2+(10µmol/L) using Benesi–Hildebrand equation.
Fig. 5 Effect of pH on probe T1 (10 µmol/L) recognition Pd2+(pH was adjusted by using Methanol-water solutions(V/V=4/6, the pH of solution was adjusted by
aqueous solution of NaOH (1 mol/L) and HCl (1 mol/L). Inset: Effect of time on probe T1 (10 µmol/L) recognition Pd2+ (λex=540 nm, λem=583nm)
Fig.6 (a) Absorbance changes of T1 (10 µmol/L) upon the addition of each equiv of EDTA with the presence of Pd2+ in methanol-water (4/6, v/v) solution. (b)
Fluorescence intensity of probe T1+Pd2+as a function of EDTA concentration in methanol-water (4/6, v/v) solution. Inset: Fluorescence intensity at 583nm for
three cycles of the switching process.
Fig.7 Job’s plot of T1 and Pd2+ (The total concentration was 20 µmol/L).
Fig. 8 Probe T1 for Pd2+ fluorescence images in HepG2 living cells. Fluorescence images of HepG2 cells treated with probes (20 µmol/L) in either the absence
(a) or the presence (c) of 20 µmol/L Pd2+ for 1h at 37 °C. (b) Bright-field image of cells shown in panel. (d) Overlay image of (b) and (c).
Scheme 1
O
COOH
N
NH2
N2H4
·H2O
ethanol , reflux
N
O
N
N
O
N
Cl
2
Rhodamine B
CHO
OHC
ethanol, r.t.
HO
NH2
O
OH
N
O
CHO
N
N
N
N
ethanol, r.t.
N
O
N
N
O
N
3
4
Scheme 2
O
Pd2+
O
N
N
N
O
N
N
Page 5 of 7

Fig. 1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
3000
2500
2000
1500
1000
500
(b)
(a)
Pd2+
Pd2+
Blank and other Ions
550
Blank and other ions
0
500
600
650
700
560
580
600
620
640
660
680
Wavelength (nm)
Wavelength ( nm)
Fig. 2
Fig. 3
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(b)
4000
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
(a)
0
0
5
10
15
20
25
30
Pd2+
[
µmol/L]
0
500
550
Wavelength (nm)
600
560
580
600
620
640
660
680
Wavelength (nm)
Fig. 4
Equation
y = a + b*x
Weight
Residual Su
Adj. R-Squar
No Weighting
0.4219
0.9954
12
10
8
Value
0.41416
1.21731E-5 2.75639E-7
Standard Err
0.14751
B
B
Intercept
Slope
y=1.217*10-5x+0.414
6
4
2
0
200000
400000
600000
800000
1000000
1/[Pd2+
]
Fig. 5
Page 6 of 7

2+
T1+ Pd
T1+ Free
4000
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
0
0
2
4
6
8
10
Time (min)
0
1
2
3
4
5
6
7
8
9
10 11 12 13
pH
Fig. 6
0.8
(b)
4500
4000
3500
3000
2500
2000
1500
1000
500
(a)
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2+
2+
T1+Pd +EDTA+Pd
2+
T1+Pd 2+
+NEt +Pd
2+
3
T1+Pd
A
C
E
G
F
D
B
O
0
T1+Free T1+Pd2++NEt3
-500
0
1
2
3
4
Cycle
T1+Pd2++EDTA
0
0.0
0.5
1.0
1.5
2.0
560
580
600
620
640
660
680
Equiv of EDTA
Wavelength(nm)
Fig. 7
3500
3000
2500
2000
1500
1000
500
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0
0.2
0.4
[Pd2+
0.6
0.8
1.0
]/
[Pd2++T]
0
0.0
0.2
0.4
[Pd2+
0.6
0.8
1.0
]/
[Pd2++Probe]
Fig. 8
Page 7 of 7