Selective and sensitive off-on probes specific for palladium detection and their application in biological environments

Article abstract of DOI:10.1016/j.jphotochem.2016.09.022

Two rhodamine B derivatives ROP and ROM have been rationally designed, synthesized and characterized. The probes exhibited highly selective Pd (II) – enhanced absorbance and fluorescence emission above 540?nm in ethanol-water (4/6, v/v, 20?mmol/L HEPES, pH 7.4) by a reversible way. Upon the addition of Pd (II), the rhodamine spirolactam ring was unfolded and a 1:1 metal-ligand was formed, which can be used for “naked-eyes” detection. In addition, cytotoxicity and bioimaging studies by HepG2 living cells and living mice indicated that the probes were negligible cytotoxicity, cell permeable and suitable for detecting Pd (II) in biological environments.

Full text of DOI:10.1016/j.jphotochem.2016.09.022

Accepted Manuscript
Title: Selective and Sensitive Off-On Probes specific for
Palladium detection and Their Application in Biological
Environments
Author: Ying Long Yin-juan Bai Jia Zhou Bing-qin Yang
PII:
S1010-6030(16)30525-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2016.09.022
JPC 10374
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Journal of Photochemistry and Photobiology A: Chemistry
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19-9-2016
21-9-2016
Please cite this article as: Ying Long, Yin-juan Bai, Jia Zhou, Bing-qin Yang, Selective
and Sensitive Off-On Probes specific for Palladium detection and Their Application in
Biological Environments, Journal of Photochemistry and Photobiology A: Chemistry
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Selective and Sensitive Off-On Probes specific for Palladium detection and Their Application in
Biological Environments
Ying Long, Yin-juan Bai, Jia Zhou, Bing-qin Yang*
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an
710127, PR China

Highlights:


Two rhodamine-based Pd2+ fluorescence probes have been designed, synthesized and characterized.
The probes exhibited highly selective Pd (II) - enhanced absorbance and fluorescence emission above 540 nm in ethanol-
water (4/6, v/v, 20mmol/L HEPES, pH 7.4) by a reversible way.


They can respond to Pd2+ directly (without supported reagents) and rapidly (completed within 1min).
The two probes were conducted to cytotoxicity and bioimaging studies by HepG2 living cells and living mice indicated that
they were low cytotoxic, cell permeable and can be used to detect Pd2+ in living systems.
Abstract: Two rhodamine B derivatives ROP and ROM have been rationally designed, synthesized and characterized. The probes
exhibited highly selective Pd (II) - enhanced absorbance and fluorescence emission above 540 nm in ethanol-water (4/6, v/v,
20mmol/L HEPES, pH 7.4) by a reversible way. Upon the addition of Pd (II), the rhodamine spirolactam ring was unfolded and a 1:1
metal-ligand was formed, which can be used for “naked-eyes” detection. In addition, cytotoxicity and bioimaging studies by HepG2
living cells and living mice indicated that the probes were negligible cytotoxicity, cell permeable and suitable for detecting Pd (II) in
biological environments.
Keywords: Rhodamine B; Palladium fluorescence probe; Cytotoxicity; Bioimaging
1. Introduction
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 [1-3]. Pd-catalyzed reactions such as Heck, Sonogashira, Buchwald–
Hartwig and Suzuki–Miyaura reactions represent powerful transformations for the synthesis of complex molecules, which played an
significant role in pharmaceuticals [4 -11]. However, their fruitful and frequent use can also generate a high level of residual palladium,
which may result in the contamination of soil and water systems [11]. And, palladium can cause severe skin and eye irritation [12]
when it is taken into human bodies via contaminated food, medicine, and water [13]. DNA, proteins, and other biomacromolecules,
such as vitamin B6, have been reported to be able to strongly bind palladium ions, which may lead to a variety of cellular dysfunction
processes [14] and therefore could cause health hazard [15-17]. Therefore, palladium detection both in the living and the environment
has attracted tremendous attention. Detecting the content of palladium in samples was usually carried out by atomic
absorption/emission spectroscopy (AAS/AES), ion-coupled plasma emission-mass spectroscopy (ICP-MS), solid-phase
microextraction high performance liquid chromatography (SPME-HPLC), and X-ray fluorescence spectroscopy (XRF) [18-19].
Although these conventional methods provide an extremely and rapidly sensitive analysis, they need sophisticated sample-pretreatment
procedures, complicated instrumentation and rigorous experimental conditions, which may restrict their extensive applications.
Fluorescent probe has become a widely used and important tool for monitoring metal ion concentration in biological samples.
Most reported fluorescence probes have a metal chelating site linked to a fluorophore, and the metal binding causes the change in
fluorescence intensity [20-25]. The development of synthetic receptors which are capable of recognising important metal ions
(especially transition-metal ions) biologically and environmentally have attracted widespread interests from biologists, chemists,

environmentalists and clinical biochemists in recent years [26–31]. Fluorescent and colorimetric chemosensors have been
increasingly applied in palladium analysis [32-34]. However, plenty of palladium detection fluorescence probes are defective: 1)
through fluorescence quenching way [35-41]; 2) via irreversible reactions and even need supported reagents [42-44 ]; 3) colorimetric
chemosensor and no fluorescence [45 ]; 4) the recognition reaction conditions are relatively strict and have a long response time [46-
47]; 5) unsuitable for bioimaging applications [42-43,48]. Therefore, nowadays, design and synthesis palladium fluorescence probes
which are capable of overcoming the above shortcomings are still have realistic significance.
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 [49]. Owing to the rhodamines
with a spirolactam scaffold, which undergoes the 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 vast chemical sensors in recent years,
and these new designed chemosensors have been quoted in recently excellent review articles [50-53]. In spirocyclic form, the sensing
event occurs in its five-membered lactam moiety. Obviously, these properties still present abundant opportunities to design new
fluorescent probes. Herein, our research work contains the design, synthesis, structure characterizations, spectra characterizations and
biological applications of fluorescent chemosensors for selective sensing of Pd2+. In our previous work [54], we reported a fluorescent
probe T1, which are successfully applied in Pd2+ detection. Based on the successful experimental results, we try to follow and expand
this research, so, here two novel rhodamine-based chemosensors ROP and ROM were prepared by three-step synthesis using
inexpensive materials. Especially, these chemosensors showed highly selective and sensitive fluorescence “turn-on” and enhanced
behaviors toward Pd2+ followed the remarkable color changes from colorless to pink, which can be used for “naked-eyes” detection.
And, these probes can give highly selective spectroscopic responses to Pd2+ over other metal ions in ethanol-water (4/6, v/v, 20mmol/L
HEPES, pH 7.4) media by reversible way. Importantly, ROP and ROM can respond to Pd2+ directly (without supported reagents) and
rapidly (completed within 1min) with high sensitivity (DL=3.49 μmol/L, 4.64 μmol/L, respective). Moreover, the two probes were
conducted to cytotoxicity and bioimaging studies by HepG2 living cells and living mice indicated that the probes were low cytotoxic,
cell permeable and can be used to detect Pd2+ in living systems.
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
ROP and ROM were dissolved in DMF-EtOH (1/40, v/v) solution in concentration of 1mmol/L as stock solutions. And then took out
quantificational ROP and ROM in different testing system. 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 on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer (at 100
1
MHz for 13C NMR and 400 MHz for H NMR). A Bruker micro TOF-Q II ESI-TOF LC/MS/MS Spectroscopy was used to perform
mass spectra. Melting points were taken on an XT-4 micromelting apparatus and uncorrected. Results of cytotoxicity were analyzed
with the Soft max pro software (version 2.2.1) in Spectra max190-Molecular Devices. And the living cells imaging were performed on
an Olympus FV1000 conf-ocal microscopy, we set the excitation wavelength as 540 nm. The living animals imaging were conducted
on IVIS DPECTRUM, with an excitation filter 550 nm and an emission range = 580-600 nm. All reagent used for synthesis were of
analytical-reagent grade and commercially available from Aldrich. Thin-layer chromatography (TLC) and column flash
chromatography was performed on silica gel GF254 and Merck silica gel (250–400 mesh ASTM), respective. And the twice-distilled
water was used throughout the experiment.
2.2 Synthetic procedures
2.2.1 Synthesis of Compound 2

We synthesized the compound 2 from Rhodamine B by the procedures published in literatures [55-58].
2.2.2 Synthesis of Compound 3
As literature [59] reported that Compound 3 was synthesized by condensing Rhodamine hydrazide with o-phthalaldehyde.
However, we have optimized the experimental conditions in order to reduce by-products and improve the conversion rate, so, instead
of refluxing Rhodamine hydrazide with o-phthalaldehyde in ethanol, we let the condensation reaction of Rhodamine hydrazide and o-
phthalaldehyde occur at room temperature. To a 250 mL round bottom flask, an excess of o-phthalaldehyde (0.804 g, 0.006 mol) was
dissolved in 20 mL ethanol, cooled with a cold water bath. Then Rhodamine hydrazide (1.369g, 0.003 mol) was dissolved in 150 mL
ethanol and added dropwise to the above 250 mL flask within 30 min with vigorous stirring, and the stirred mixture was allowed to
stand in the cold water bath for about 4 h, then the solvent was removed under reduced pressure, further purified the resulting
precipitate by silica gel column chromatography to give 3 (light yellow solid) in 91.6% yield. MS Calcd for (C36H36N4O3) m/z =
572.2787, Found: 573.2864 (M+H)+. IR (KBr) υ: 1750, 1690, 1635, 1517, 1306, 1271, 1239, 1213, 1118, 818, 786, 756, 699 cm-1. 1H
NMR (400 MHz, CDCl3, TMS), δ (ppm): 1.13 (12H, t, J = 8 Hz), 3.27~3.33 (8H, m), 6.32~6.41 (2H, m), 6.57~6.62 (4H, m), 7.04 (1H,
d, J = 8 Hz), 7.32 (1H, t, J = 8 Hz), 7.49~7.53 (3H, m), 7.68 (1H, d, J = 8 Hz), 7.96 (1H, d, J = 8 Hz), 8.12 (1H, d, J = 8 Hz), 9.13 (1H,
s), 9.78 (1H, s).Anal. calcd. for C36H36N4O3 (%): C, 75.5; H, 6.34; N, 9.78. Found: C, 75.7; H, 6.39; N, 9.81.
2.2.3 Synthesis of Compound ROP and ROM
Synthesis of Compound ROP: Compound 3 (0.572 g, 0.001mol) was dissolved in 20ml ethanol. Then a solution of 2-
aminophenol (0.131g, 0.0012mol, a little excess) in ethanol (20 mL) was added and the mixture was stirred for 8 h at ambient
temperature. Then filtered the precipitate produced and washed 3 times with 10 mL cold ethanol. After drying under reduced pressure,
the crude product was purified by recrystallization in EtOH/H2O to give Compound ROP (light yellow solid) in 83.4% yield. mp:
188~190 °C. MS Calcd for (C42H41N5O3) m/z = 663.3209, Found: 664.3294 (M+H)+ . IR (KBr) υ: 3388, 2969, 1698, 1616,1546,
1513, 1423, 1326, 1268, 1222, 1120, 1074, 1018, 979, 869, 819, 786, 757, 719, 646, 534, 472 cm-1.1H NMR (400 MHz, CDCl3, TMS),
δ (ppm): 1.13 (12H, t, J = 8 Hz), 3.31~3.37 (8H, m), 6.22~6.24 (2H, m), 6.44~6.47 (4H, m), 7.03~7.05 (1H, m), 7.34~7.37 (1H, m),
7.39~7.486 (4H, m), 7.56~7.61 (1H, m), 7.83~8.16 (4H, m), 8.04 (1H, s), 10.75 (1H, s). 13C NMR (100 MHz, CDCl3, TMS), δ (ppm):
12.6, 44.3, 55.1, 66.0, 97.9, 98.9, 103.9, 104.9, 107.9, 123.2, 127.7, 133.9, 143.4, 149.0, 152.1, 166.5. Anal. calcd for C42H41N5O3 (%):
C, 75.99; H, 6.23; N, 10.55. Found: C, 76.02; H, 6.22; N, 10.57.
Synthesis of Compound ROM: Compound 3 (0.572 g, 0.001mol) was dissolved in 20mL methanol. Then a solution of o-
aminoanisole (0.148g, 0.0012mol, a little excess) in ethanol (20 mL) was added and the mixture was stirred for 8 h at room
temperature. Then filtered the precipitate produced and washed 3 times with 10 mL cold ethanol. After drying under reduced pressure,
The crude product was purified by recrystallization in EtOH/H2O to give Compound ROM (pale yellow powder) in 80.6% yield. mp:
113~115 °C. IR (KBr) υ: 3437, 2972, 2927, 2873, 1727, 1697, 1613, 1518, 1464, 1419, 1301, 1266, 1222, 1118, 1078, 1020, 975,
1
818, 783, 759, 695, 530 cm-1. MS calcd for (C43H43N5O3) m/z = 677.3366, Found: 678.3428 (M+H)+. H NMR (400 MHz, CDCl3), δ
(ppm): 1.17 (12H, t, J = 8 Hz), 3.33~3.39 (8H, m), 3.75 (3H, s), 6.23~6.26 (2H, m), 6.43~6.49 (4H, m), 7.04 (1H, d, J = 4 Hz),
7.37~7.39 (1H, m), 7.42~7.49 (4H, m), 7.59 (1H, d, J = 4 Hz), 7.86 (1H, d, J = 4 Hz), 7.95 (1H, d, J = 4 Hz), 8.05 (1H, d, J = 4 Hz),
8.17 (1H, d, J = 8 Hz), 9.56 (1H, s), 10.78 (1H, s). 13C NMR (100MHz, CDCl3), δ (ppm): 12.6, 44.3, 55.9, 66.4, 97.9, 105.9, 115.1,
115.9, 120.1, 122.7, 129.0, 133.5, 135.3, 135.9, 136.2, 145.9, 148.9, 151.5, 156.6, 165.0. Anal. calcd for C43H43N5O3 (%): C, 76.19; H,
6.39; N, 10.33. Found: C, 76.20; H, 6.38; N, 10.35.
2.3 Preparation of the test solution
The 10 μmol/L test solution of probes ROP and ROM were prepared in ethanol-water (4/6, v/v, 20mmol/L HEPES, pH 7.4). 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, PtCl2, BaCl2·2H2O, NaCl, AlCl3·6H2O and FeCl3·6H2O dissolved in
the double distilled water. Before spectroscopic measurements, the corresponding solutions of probes were freshly prepared by diluting
the high concentration stock solutions. All the measurements were made according to the procedures as follows. Placing 1 mL of the
probe solution and an appropriate aliquot of each metal stock into a 10 mL glass tube, and diluting the solution to 10 mL with ethanol-
water (4/6, v/v, 20mmol/L HEPES, pH 7.4) solution. The absorbance was showed at 550 nm and the fluorescence emission appeared at
583 nm. Both the excitation and emission wavelength band passes were set as 5.0 nm and the excitation wavelength was set at 540 nm.
2.4 Cytotoxicity assay
The MTT (5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) assay was performed to evaluate the toxicity of ROP, ROM
and Pd2+ by HepG2 cells [60]. 90% confluent cell were chosen, digested by 1 mL 0.25% of trypsin, and transferred in 96-well plates.
The cells were treated and incubated at 37 °C under 5% CO2 in culture medium (RPMI 1640 + 10% FBS) and maintained 24 h.
Different concentrations of ROP, ROM and Pd2+ were added to the 96-well plates, respectively. Then another 24 h incubation was
taken at the same condition. Following this, the medium was removed and washed three times with phosphate buffered saline (PBS).
Then the medium was replaced with mixed liquor of MTT（5 mg/mL）and culture medium and incubated for an additional 4 h. After
that, the MTT was removed and washed three times with PBS. Subsequently, 150 μL DMSO was carefully added to each well and
ultrasonic oscillation for 10 minutes. All the experiments were conducted in triplicate. The cell viability (%) was calculated according
to the Equation: Cell viability%= [OD490 (sample)/ OD490 (control)] ×100%, where OD490 (sample) represents the optical density of
the wells treated with various concentration of probes or Pd2+ and OD490 (control) represents that of the wells treated with ethanol.
2.5 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 processed with probes ROP and ROM (10 μmol/L) for 1 h at 37 °C 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 °C, cells were washed 3 times with PBS to remove
remaining Pd2+ and then imaged. Confocal fluorescence imaging was carried out with an Olympus FluoView FV1000 laser scanning
microscope with 80× objective lens. Excitation of probe-loaded cells at 540 nm was performed with a solid laser and emission was
collected at 550–750 nm.
2.6 Fluorescence imaging in living animals
Kun Ming mice (15-20g) were anesthetized by an injection of xylazine (10 mg/kg) and ketamine (80 mg/kg). Then, the mice were
given an intraperitoneal (10 μmol/L, 5μL) injection of the probes ROP and ROM. After ten minutes, The mice were injected with Pd2+
(10 μmol/L, 5μL) and then imaged by using an IVIS imaging system with excitation and emission filters set at 550 nm and 580nm,
respectively.
3. Results and discussion
3.1 Spectroscopic properties
The probes ROP and ROM exhibited prominent selective and sensitive response to Pd2+. Hydroxy group and methoxy group
moieties have effects on the fluorescence intensities. However, no obvious differences were found on the other spectral properties for
the two probes. Thus, ROP was chosen for further discussion, optical spectra of ROM was shown in the Supporting Information.
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 ROP in the presence of Pd2+ and other competitive metal
ions were recorded in Fig.1 a and b, respectively. There was no obvious fluorescence changes were observed in the presence of other
cations (Fig.1c), such as heavy and transition metal ions (Mn2+, Pb2+, Cd2+, Co2+, Fe3+, Cu2+, Ni2+, Ag+, Zn2+, Cr3+, Pt2+ , Hg2+), alkali or
alkaline-earth metals (Na+, Ca2+,Mg2+, Ba2+) and Al3+.

However, the F/F0 value was more than 100-fold increase 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
ROP+Pd2+ and ROM+Pd2+complex could be conveniently distinguished from those of the free ROP and ROM by the naked eye, as
shown in Fig.2. This strongly suggested ROP and ROM has high selectivity to divalent palladium ion and can serve as “naked- eyes”
probes for Pd2+.
Fluorescence and UV titration experiments were carried out for the sake of further investigating the interaction of ROP and Pd2+.
Since the stable “spirolactam form” of rhodamine, free ROP represents colorless and no fluorescence response in the range from 550
to 750 nm. However, upon gradual addition of Pd2+, significant enhancement of fluorescence with an emission maximum at 583 nm
(Fig.3a) and an absorbance maximum at 550 nm (Fig.3b) were 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 adding more than 1.0 equiv of Pd2+, the fluorescence intensity showed negligible changes (Fig. 3a, inset). The results clearly
indicated that, owing to the addition of Pd2+ to ROP, the spirolactam form was unfolded, and formed a highly delocalized π-conjugated
structure. Thus, obvious and significant enhancement of absorbance and fluorescence were observed.
The association constant K was calculated according to the Benesi–Hildebrand equation [61] : (Fmax − F0)/(Fx − F0) = 1 + (1/K)
(1/[Pd2+]), where Fmax, F0 and Fx are fluorescence intensities of 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= 2.45 × 104 L/mol. The linearity of this plot
ensures the formation of a 1:1 clathrate between ROP and Pd2+ (R=0.99338). When the fluorescence intensity reached the maximum of
the probe ROP to Pd2+，F/F0 was as high as over 100-fold, demonstrating that ROP has the excellent capability of detecting Pd2+ both
quantitatively and qualitatively.
It is well known that the spirolactam ring of the rhodamine derivative is 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 ROP. The effects of pH were
evaluated in the pH range from 2.0 to 12.0 (Fig. 5a). 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 was formed under different pH values. It showed the pH value corresponding to the highest
response approximately in 5~10, which revealed that the probe ROP for Pd2+ could work well in approximate physiological conditions
with a very low background response. Therefore, further studies were carried out in ethanol-water (4/6, v/v) at pH 7.4. Furthermore,
the time-dependence of probe ROP fluorescence was also evaluated in the presence of Pd2+ ions. The results showed that, upon the
reaction of probe ROP with Pd2+, the fluorescence of all the tested solutions remarkably increased to their maximum value within the
first 1 min. (Fig.5b).
3.2 Calculation of detection limit
The detection limit of ROP and ROM were determined from fluorescence titration data (Fig.3a, Fig.S2a) based on reported and
broadly used method [62-64]: According to the results of titration experiments, graphs of (Fmin - F) / (Fmin - Fmax) versus log
[Pd2+] were plotted, where the F is the fluorescence intensity at 583 nm at each concentration of Pd2+ added, Fmin and Fmax are
the minimum and maximum fluorescence intensity at 583 nm respectively. Linear regression curves were then fitted (Fig.6), and

the intercepts of the lines at x-axis were taken as detection limit of ROP (3.49 μmol/L) and ROM (4.64 μmol/L).
3.3 Reversibility and proposed mechanism for the binding of probes with Pd2+
In this section, we only discuss the experimental results of ROP interacted with Pd2+, the experimental results of ROM are
depicted in Supporting Information.
As is well known, the reversibility is an important property to obtain an excellent probe. Thus, the EDTA–adding experiments
were conducted to examine the reversibility of the probes ROP. As clearly shown in Fig.7a, the absorbance decreased when EDTA
was added to the mixture containing ROP and Pd2+. Besides, the color of mixture changed back to colorless and fluorescence intensity
decreased. When Pd2+ was added to the system again, the signals were almost completely reproduced (Fig. 7b) and the colorless
solution turned to pink. The above process can be repeated several cycles without significant changes in the fluorescence spectrum
(Fig.7b.insert). These findings indicated that probe ROP is a reversible fluorescent probe toward Pd2+.
Binding analysis using the method of continuous variations (Job’s plot) was measured. A maximum absorbance at 550 nm
(Fig.8.insert) and fluorescence emission at 583 nm (Fig.8) were observed when the molecular fraction of ROP are close to 0.5, which
established the 1:1 complex formation between ROP 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. And proposed binding modes between the probes
ROP&ROM and Pd2+ are shown as Scheme 2. It is very likely due to the chelation–induced ring opening of rhodamine spirolactam,
rather than other possible reactions [51, 65].
3.4 Cytotoxicity assays and biological imaging applications
To ascertain the cytotoxic effects of ROP, ROM and Pd2+, the MTT assay was performed according to the reported method
[60].The details of MTT experimental operations can be seen in Experimental section of this article. Data are expressed as mean ±
standard deviation in Table 1 (supporting information).The results were illustrated in Fig.9. The cell viability still remains 99.71%,
91.06%, 92.92% under the treatment of 25 μmol/L probes and Pd2+, which indicated that ROP, ROM and Pd2+ are low cytotoxic to
cells and suitable for bioimaging.
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. 10. In
the absence of Pd2+, free ROP and ROM showed no detectable fluorescence signal in living cells (Fig. 10a). By contrast, after
incubation with Pd2+, a bright fluorescence was observed in living cells (Fig. 10c). Bright-field transmission images of cells treated
with Pd2+ and probe revealed that the cells were viable throughout the imaging experiments (Fig. 10b). The results suggested that
probes ROP and ROM can penetrate the cell membrane and can be applied for in vitro imaging of Pd2+ in living cells and potentially
in vivo.
Further research on the potential of biological imaging applications, fluorescence imaging in living mice was performed [66]. The
probes ROP and ROM (10 μmol/L，5μL) was injected into the living mice and no fluorescence signal were noted (Fig .11a). After

ten minutes, as the Pd2+ injection, a large fluorescence signal was observed within a few minutes of injection (Fig .11b).This indicates
the potential in vivo imaging applications of the probes.
4. Conclusion
In summary, we have described two novel rhodamine-based probes ROP and ROM, they can give selective and sensitive
fluorescence enhancement response to Pd2+ via a reversible sensing mechanism in ethanol - water solution. High selectivity toward Pd2+
is exhibited and little cross-sensitivity is observed to other commonly metal ions. The molecular design might greatly contribute to the
development of more efficient and useful probes based on rhodamine platform. The excellent biological values of probes are
demonstrated by the cytotoxicity and fluorescence imaging in HepG2 living cells and living mice. It is anticipated that the probes will
significantly promote 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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3000
2500
2000
1500
1000
500
（ a）
2+
Pd
2+
2+
2+
Mg , Cd , Mn
,
2+ 2+ 2+
Ni , Ba , Zn
3+ 2+
,
+
Al , Na , Ca
2+
,
+
2+
Ag , Hg , Cu
,
3+
2+
3+
Fe , Co , Cr
2+ 2+
,
Pb , Pt , only ROP
0
560
580
600
620
640
660
wavelength (nm)
（ b）
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2+
Pd
2+
2+
2+
Mg , Cd , Mn
,
2+ 2+ 2+
Ni , Ba , Zn
3+ 2+
,
+
Al , Na , Ca
2+
,
+
2+
Ag , Hg , Cu
,
3+
2+
3+
Fe , Co , Cr
2+ 2+
,
Pb , Pt , only ROP
500
550
600
Wavelength（ nm）

3500
3000
2500
2000
1500
1000
500
2+
ROP+ions+Pd
ROP+ions
(c)
0
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Fig.1 Fluorescence (a) and absorbance (b) spectra of ROP (10 μmol/L) in ethanol-water (4/6, v/v, HEPES=20mmol/L, pH=7.4) solution upon addition of
various metal ions (10μmol/L), λex=540 nm. (c) Fluorescence intensity changes of ROP (10μmol/L) upon the addition of various metal ions (10μmol/L) in the
presence of Pd2+ (10μmol/L) in ethanol-water (4/6, v/v, HEPES=20mmol/L, pH=7.4) solution. The black bars represent the fluorescence response of ROP 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.Pt2+,
18.Pd2+. The red bars represent the subsequent addition of 10μmol/L Pd2+ to the above solutions. Error bar=RSD (n=3)

Fig.2 (a) and (c) the color change of the probes ROP and ROM in different metal ions(1. Na+，2. Al3+, 3.Ca2+, 4.Mg2+, 5.Cd2+, 6.Mn2+, 7.Ni2+, 8.Ba2+, 9. Pd2+,
10. Cr3+ 11.Co2+, 12. Pt2+, 13.Pb2+, 14.Ag+, 15.Fe3+, 16.Hg2+, 17.Cu2+, 18. Zn2+); (b) and (d) the fluorescence change of the probes ROP and ROM in different
metal ions( both 10μmmol/L)

(a)
3000
2500
2000
1500
1000
500
3000
2500
2000
1500
1000
500
0
0
2
4
6
8
10
12
14
[Pd2+
]
(μmol/L)
0
560
580
600
620
640
660
wavelength (nm)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(b)
450
500
550
600
650
Wavelength (nm)
Fig.3 (a) Fluorescence intensity changes of ROP (10μmol/L) upon addition of Pd2+ in ethanol-water (4/6, v/v, HEPES=20mmol/L, pH=7.4) solution. Inset:
Changes in the emission intensity at 583nm. (b) Absorbance intensity changers of ROP (10μmol/L) upon addition of Pd2+ in ethanol-water (4/6, v/v,
HEPES=20mmol/L, pH=7.4) solution.

4
y=4.07507*10-5X-1.1247
R=0.99338
2
0
40000
60000
80000
100000
1/[Pd2+] （ L/mol）
Fig.4 Determination of binding constant of ROP (10 μmol/L) with Pd2+ (10 μmol/L) using Benesi–Hildebrand equation.

ROP+Pd2+
only ROP
3000
2500
2000
1500
1000
500
only ROP
ROP+Pd2+
3000
2500
2000
1500
1000
500
（ a）
(b)
0
2
4
6
8
10
12
0
2
4
6
pH
Time(min)
Fig 5 (a) Effect of pH on ROP (10 μmol/L) recognition Pd2+ (the pH of solution was adjusted by aqueous solution of NaOH (1mol/L) and HCl (1mol/L). (b)
Effect of time on probe ROP (10 μmol/L) recognition Pd2+ (λex=540 nm, λem=583nm)

1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
1.2
0.6
0.0
（ a）
（ b）
y=5.95372+1.09091x
R=0.99934
y=8.68304+1.62799x
R=0.99872
-5.5
-5.4
-5.3
-5.2
-5.1
-5.0
-4.9
-4.8
-4.7
-5.4
-5.3
-5.2
-5.1
-5.0
-4.9
-4.8
-4.7
2+
2+
log[Pd
log[Pd
]
]
Fig.6. The plots of (Fmin - F) / (Fmin - Fmax) versus log [Pd2+] for the probes ROP (a) and ROM (b).

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2+
(a)
2+
(b)
ROP+Pd +EDTA+Pd
3000
2500
2000
1500
1000
500
3000
2500
2000
1500
1000
500
2+
2+
2+
ROP+Pd +EDTA+Pd
ROP+Pd
2+
0
ROP+Pd +EDTA
only ROP
2+
ROP+Pd +EDTA
0
2
4
6
8
10
cycle
0
560
580
600
620
640
660
0.0
0.5
1.0
1.5
2.0
Wavelength (nm)
Equiv of EDTA
Fig.7 (a) Absorbance intensity changes at 550 nm of ROP (10 μmol/L) upon the addition of each equiv of EDTA with the presence of Pd2+ in ethanol-water
(4/6, v/v, HEPES=20mmol/L, pH=7.4) solution. (b) Fluorescence intensity of probe ROP+Pd2+as a function of EDTA concentration in ethanol-water (4/6, v/v,
HEPES=20mmol/L, pH=7.4) solution. Inset: fluorescence intensity at 583nm for four cycles of the switching process.

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
0.6
0.8
1.0
[Pd2+]/[Pd2++ROP]
0
0.0
0.2
0.4
0.6
0.8
1.0
[Pd2+]/[Pd2++ROP]
Fig.8 Job’s plot of ROP and Pd2+ (The total concentration was 10 μmol/L).

ROP
110
105
100
95
ROM
2+
Pd
90
85
80
75
0
20
40
60
80
100
Probes & Pd2+ (umol/L)
Fig. 9 HepG2 living cells viability of ROP, ROM and Pd2+ were quantified by the MTT assay (mean ± SD).

Fig.10 Probe ROP and ROM for Pd2+ fluorescence images in HepG2 living cells. Fluorescence images of HepG2 cells treated with probes (10 μmol/L) in
either the absence (a) or the presence (c) of 10 μmol/L Pd2+ for 1h at 37 °C. (b) Bright-field image of cells shown in panel. (d) Overlay image of (b) and (c).

Fig. 11 Representative fluorescence images for the mouse. ( (a).only injection with 10 μmol/L, 5 μL ROP and ROM and (b) .after injection with 10 μmol/L, 5
μL Pd2+.)The mice were imaged using IVIS DPECTRUM, with an excitation filter 550 nm and an emission range = 580-600 nm.

O
COOH
N
N2H4
.
H2O
N
NH2
ethanol reflux
N
O
Cl-
N
O
2
N
Rodamine
B
CHO
CHO
R
N
ethanol RT
OHC
O
NH2
R
O
N
N
N
N
ethanol RT
N
O
ROP&ROM
N
N
O
3
N
ROP: R= -OH; ROM: R= -OCH3;
Scheme 1 Synthesis of ROP and ROM

Scheme 2 The most likely binding modes between the probes ROP&ROM and Pd2+