An umbelliferone-derivated fluorescent sensor for selective detection of palladium(II)from palladium(0)in living cells

Article abstract of DOI:10.1016/j.saa.2019.05.039

Palladium (Pd)has drawn worldwide attentions because its connections to industry, chemistry, biological material and public health. Quantitative and selective detection tools for Pd and its ion forms are in urgent necessity. Here an umbelliferone derivative Umb-Pd2 was provided as a small, steady, safe and selective sensor for detecting Pd(II). It indicated advantages including sensitive (LOD 1.1 nM), wide pH tolerance (5.0–10.0), applicable linear range (0–1.8 equivalent)and low toxicity. The most attractive point was its explicit selectivity towards Pd(II)from Pd(0)in both independent and coexistence systems. This distinguishing ability was further utilized in imaging in living cells, raising this work as a rare and important example among all the published papers on palladium sensing. Thus, Umb-Pd2 supplied a potential approach for further improvement and applications in both daily chemistry and public health.

Full text of DOI:10.1016/j.saa.2019.05.039

Accepted Manuscript
An umbelliferone-derivated fluorescent sensor for selective
detection of palladium(II) from palladium(0) in living cells
Xu-Ping Zhang, Qing Yuan, Ya-Lin Qi, Da-Jun Zheng, Qi-Xing
Liu, Bao-Zhong Wang, Yu-Shun Yang, Hai-Liang Zhu
PII:
DOI:
Reference:
S1386-1425(19)30521-9
https://doi.org/10.1016/j.saa.2019.05.039
SAA 17134
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 15 April 2019
Revised date:
Accepted
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5 May 2019
14 May 2019
Please cite this article as: X.-P. Zhang, Q. Yuan, Y.-L. Qi, et al., An umbelliferone-
derivated fluorescent sensor for selective detection of palladium(II) from palladium(0)
in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
https://doi.org/10.1016/j.saa.2019.05.039
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ACCEPTED MANUSCRIPT
An umbelliferone-derivated fluorescent sensor for selective
detection of palladium(II) from palladium(0) in living cells
Xu-Ping Zhang‡, Qing Yuan‡, Ya-Lin Qi, Da-Jun Zheng, Qi-Xing Liu, Bao-Zhong Wang*,
Yu-Shun Yang* and Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing
University, Nanjing, 210023, China.
*Corresponding authors. Tel. & Fax: +86-25-89682572; E-mail: zhuhl@nju.edu.cn;
ys_yang@nju.edu.cn; wbz@nju.edu.cn.
‡Both authors contributed equally to the work
ABSTRACT: Palladium (Pd) has drawn worldwide attentions because its connections to
industry, chemistry, biological material and public health. Quantitative and selective detection
tools for Pd and its ion forms are in urgent necessity. Here an umbelliferone derivative
Umb-Pd2 was provided as a small, steady, safe and selective sensor for detecting Pd(II). It
indicated advantages including sensitive (LOD 1.1 nM), wide pH tolerance (5.0 - 10.0),
applicable linear range (0 - 1.8 equivalent) and low toxicity. The most attractive point was its
explicit selectivity towards Pd(II) from Pd(0) in both independent and coexistence systems.
This distinguishing ability was further utilized in imaging in living cells, raising this work as a
rare and important example among all the published papers on palladium sensing. Thus,
Umb-Pd2 supplied a potential approach for further improvement and applications in both
daily chemistry and public health.
Keywords: Palladium, Fluorescent sensor, High selectivity, Biological imaging, Umbelliferone
derivative.
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1. Introduction
Palladium (Pd) has drawn worldwide attentions of the common population with its
impressive functions and risks along with the series movies "Iron Man" [1]. Since science and
technology nowadays are stepping forward and indistinguishing the boundary between the real
world and future science, the researches have been required to be more prospective [2]. Even
in the daily life, Pd is an essential rare metal with widely known industial ultilities in
producing electrical contracts, spark plugs, fuel cells and catalytic converters [3,4]. Especially
for the chemists, its high selectivity and activity helped catalyze dozens of reactions incuding
Buchwald-Hartwig, Heck, Suzuki-Miyaura, Songashira reactions and so on [5-8]. Meanwhile,
more connections have been built between Pd and public health due to its use in
pharmaceutical synthesis and detrimental effect on biomolecules including DNA, amino acids
and proteins [9-11]. The daily dietary intake of Pd should be limited within 1.5 - 15 μg [12,13].
Thus, it has become an increasing emergency to develop sensitive and selective aproaches on
detecting Pd in the environment and living organisms [14,15]. The available methods include
inductively coupled plasma mass spectroscopy, atomic absorption spectrometry and high
performance liquid chromatography [16-19]. However, typical disadvantages such as complex
preparations, high-cost equipments and expereienced operaters block their convenient
applications [20,21]. In response to the requirements of the times, various fluorescent probes
for detecting Pd and its ionic forms have been rapidly developed [22-57], yet two major
concerns seem to block their further applications. One is the biocompatibility, such as safety
and solubility [58-60]. The other is the distinguishing ability between Pd(0) and Pd(II).
Though many researchers designed their probes for only one form, actually the selectivity
between these two forms is still seldom reported.
In this work, we provided an umbelliferone derivative Umb-Pd2 ("Umb" for the
structure umbelliferone and "Pd2" for Pd(II)) as a small, steady, safe and selective sensor for
detecting Pd(II) (shown in Figure 1). The corresponding detecting product via reation with
Pd(II) was named Umb-Fl ("Umb" for the structure umbelliferone and "Fl" for fluorescence
turnig-on). The original umbelliferone structure is from natural product, convinced safty in
pharmacal and biological uses [61-63]. Despite the effect as an inhibitor [64], it was the first
time to use Umb-Pd2 as a sensor. Moreover, the biocompatibility has also been evaluated.
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The most attractive point was its selectivity towards Pd(II) from Pd(0), which might be
essential for precise detection in both biological and catalytic systems. This feature was quite
distinguishing among the investigations claiming selectivity for Pd(0). The imaging in living
cells was also conducted to reveal the potential for future applications.
Figure 1. Shematic illustration of selective detection for Pd(II) by Umb-Pd2.
2. Materials and methods
2.1. General methods
The commercially available chemicals were directly used without purification. Bruker
1
DRX-600 spectrometer was used for H NMR and 13C NMR spectra. NMR data were resolved
with MestreNova software. Agilent 6540 UHD Accurate Mass Q-TOF LC/MS was recruited for
Mass spectra. PHS-25 pH-meter was used for pH measurement. UV-vis spectra were recorded on
Shimadzu UV-2550 spectrometer. Fluorescence measurements were obtained from Hitachi
Fluorescence Spectrophotometer F-7000. Cell viability were performed using HeLa (human
cancer cell line) and HEK293T (human embryonic kidney cell line) by MTT method. The imaging
experiments were performed using single-photon confocal fluorescent microscope (Carl Zeiss
LSM 880). HeLa cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with
10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin, incubated in an atmosphere
containing 5% CO2 for 12 h before imaging.
The chloride salt dissolved in distilled water for metal ions (2.0 mM) except Pb2+ (from
Pb(Ac)2), Ag+ (from AgNO3) and Pd2+ (from Pd(Ac)2). Pd(0) was from Pd(PPh3)4. Umb-Pd2 was
dissolved in MeCN to get the solution of 1.0 mM. Other concentrations were obtained by dilution.
In the selectivity evaluation, Umb-Pd2 was set as 10 μM with 10-fold (100 μM) of Pd(II) and
100-fold (1 mM) of other analytes. The detection systems were incubated for 30 min at 37 ℃. The
excitation wavelength was 322 nm. Both excitation and emission slit widths were 5 nm. The
photomultiplier voltage was 400 V.
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2.2. Determination of the fluorescence quantum yield
2
Participation ratio method was recruited with formula: Φu = [(AsFun2)/ (AuFsn0 )]Φs.
Φu are the determined fluorescence quantum yields of the tested samples. Φs is the quantum
yield of the reference substance. As and Au represent the absorbance of the reference and tested
samples at the excitation wavelength, respectively. Fs and Fu refer to the integrated emission band
areas under the same conditions, respectively. n and n0 are the solvent refractive indexes of
determined and reference, respectively.
Herein, quinine sulfate was used as control, Φs = 0.56, As = 0.008, Fs = 80.441, n0 = 1.333;
For the sensor Umb-Pd2: Au = 0.027, Fu = 174.16, n = 1.343; Φu = 0.36;
For the detecting product Umb-Fl: Au = 0.018, Fu = 209.00, n = 1.343; Φu = 0.66.
2.3. Ethics statement
These experiments were conducted in accordance with the guideline issued by the China
Food and Drug Administration (CFDA of China). Cell lines were cultured and passaged in
accordance with the guidelines established by the National Science Council of Republic China. All
experimental protocols were approved by Academic Committee of Nanjing University.
2.4. Synthesis of Umb-Pd2
The general synthesis route of Umb-Pd2 was shown in Figure 2. 4-Methylumbelliferone
(1.762 g, 10.0 mmol) was dissolved in 25 mL acetone in a dry round flask. 3-Bromo-1-propyne
(0.825 mL, 11.0 mmol) and K2CO3 (2.070 g, 15.0 mmol) were added. The reaction mixture was
refluxed for 18 h and monitored by TLC. After finishing the reaction, the solvent was removed in
vacuo and the raw product was purified by silica gel column chromatography (PE:EA = 4:1) to
give Umb-Pd2 as a white solid (851 mg, yield 56 %). 1H NMR (600 MHz, DMSO-d6) δ 7.70 (d,
J = 8.8 Hz, 1H), 7.05-6.99 (m, 2H), 6.24 (s, 1H), 4.95 (d, J = 2.2 Hz, 2H), 3.67 (s, 1H), 2.40 (s,
3H). 13C NMR (150 MHz, DMSO-d6) δ 160.5, 154.94, 153.78, 126.97, 114.13, 113.00, 111.99,
102.28, 79.37, 79.00, 56.55, 18.60. HRMS (ESI-TOF m/z): Calculated for [C13H11O3]+: 215.0708,
Found: 215.0711.
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Figure 2. General synthesis route of Umb-Pd2.
3. Results and discussion
3.1. Synthesis of Umb-Pd2
Umb-Pd2 was synthesized as shown in Figure 2 and confirmed by satisfactory spectroscopic
data (1H NMR, 13C NMR and HRMS, Figure S7-9, SI).
3.2. General fluorescent properties and sensitivity for sensing Pd(II)
The designing concept of Umb-Pd2 was introducing a typical recognizing group onto a
suitable fluorophore thus presenting a ratiometric fluorescence change when being triggered. One
key point was employing the boundary between ultraviolet and visible light to generate a turn-on
phenomena in biological imaging. After recruiting the umbelliferone structure, we initially
evaluated the general fluorescent properties of Umb-Pd2. The absorption spectrum of
Umb-Pd2 and the detecting product Umb-Fl were scanned (Figure S1, SI). When excited at 322
nm, their emission bands were at 380 nm (Umb-Pd2) and 450 nm (Umb-Fl), respectively. Their
fluorescence quantum yields were 0.36 (Umb-Pd2) and 0.66 (Umb-Fl), respectively. By using the
detecting system containing Umb-Pd2 (10 μM) and Pd(II) (100 μM) in PBS buffer (pH 7.4, 10
mM, 10% MeCN v/v) at 37 ℃, the fluorescence signals were steady in the pH range of 5.0-10.0,
which was a relatively wide window for application (Figure S2, SI). Meanwhile, the reaction time
was determined as 30 min and the fluorescence could retain for over 24 h (Figure S3, SI). Thus it
was a relatively rapid and steady system.
The fluorescence spectrum of Umb-Pd2 (10 μM) with increasing concentrations of added Pd
(II) in Figure 3a indicated a ratiometric fluorescence change from 380 nm to 450 nm. As shown in
Figure 3b, the plateau was at 10.0 equivalent and the linear range was 0 - 1.8 equivalent. The limit
of detection (LOD) was then determined to be 1.1 nM (using the formula 3σ/k, σ meant the
standard deviation with 0 equivalent Pd(II) and k meant the slope of the linear regression curve).
All these results guaranteed the potential of Umb-Pd2 in applications of imaging in living cells
and testing in biological fluid.
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Figure 3. (a) The fluorescence emission spectra of Umb-Pd2 upon increasing concentrations of
Pd(II) (0 - 20 equiv.); (b) The logistic fitting in the range of 0 - 20 equivalent; (b-Inner) The linear
correlation in the range of 0 - 1.8 equivalent.
3.3. Selectivity of Umb-Pd2 towards Pd(II)
The selectivity of Umb-Pd2 towards Pd(II) was evaluated with various analytes (Figure 4a).
No significant response was observed except Pd(II). Though many researchers claimed that their
probes were for either Pd(II) or Pd(0), explicit reports on this kind of selectivity was rare and
mainly for Pd(0) [22,25,51]. Here, Umb-Pd2 could selectively detect Pd(II) from Pd(0). The
broadened selectivity was tested using anions (Figure S4, SI) and amino acids (Figure S5, SI). Still,
Umb-Pd2 showed superior specifity for Pd(II). Moreover, the competitive experiment was
conducted (Figure 4b) to further confirm the performance of Umb-Pd2. The coexistence of other
analytes did not interfere the response of Umb-Pd2 on Pd(II). This capabillity was significant
for developing precise quantitive tools in both biological and catalytic systems.
Figure 4. The intensity ratio (I450/I380) values indicated the selectivity of Umb-Pd2 towards
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Pd(II) (a) from other analytes and (b) in the coexistence systems.
3.4. Proposed mechanism
The recognition mechanism could be rationaly explained as a typical catalytic process of
1
Pd(II) with the propargyl group (Figure 5a). The H NMR data of Umb-Pd2, Umb-Pd2 with
Pd(II) (separated), Umb-Pd2 with Pd(0) (separated) and Umb-Fl (Figure 5b) also supported this
mechanism. As seen in Figure 5c, the HRMS (ESI-TOF) data also agreed with the above
mentioned mechanism with the correct molecular weights. After incubated with Pd(II), the peaks
of the propargyl group disappeared and the hydroxyl group could be observed. Moreover, an
extra benefit was that the sensor Umb-Pd2 could be regenerated from the detecting product
Umb-Fl. Concerning both the pre-clinical potential and industrial use, since Pd could not easily
circulate in the environmental system, this kind of recycling was favorable for both economic and
environmental considerations.
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Figure 5. (a) The Pd(II)-induced depropargylation reaction of Umb-Pd2, (b) the 1H NMR data
and (c) the HRMS data comparison of corresponding compounds.
3.5. Imaging in living cells and application in biological fluids
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Before imaging in living cells, the cytoxicity of Umb-Pd2 was evaluated on HeLa and
HEK293T cells (Figure S6, SI). With detecting (10 μM) and higher concentrations (20, 50 μM) of
Umb-Pd2, over 80% of the cells remained alive, indicating that Umb-Pd2 was low toxic.
Subsequent confocol experiment in HeLa cells was carried out to evaluate the cell
permeability and imaging capability of Umb-Pd2 (Figure 6). The cells were incubated with
Umb-Pd2 (10 μM) for 30 min at 37 ℃. Neither treated with buffer (Figure 6a-c) nor treated with
100 μM Pd(0) (Figure 6d-f) for another 30 min could generate fluorescence signals in the blue
channel. However, when incubated with Pd(II) (Figure 6g-i, j-l) for another 30 min, strong blue
fluorescence could be observed with the increasing concentrations (50, 100 μM). This result
agreed with the designing concept of utilizing the boundary of ultraviolet and visible light to
generate a turn-on phenomena. Thus, Umb-Pd2 could be employed to monitor the concentrations
of Pd(II) in living cells with applicative cell permeating ability.
The practical application of Umb-Pd2 to test added Pd(II) in rat urine samples was then
performed. After the dilution by 100 times to eliminate the background noise, the addition of Pd(II)
with known concentrations was conducted. The recovery varied in the range of 98.40 - 103.7 %
(Table S1, SI), inferring the potential of Umb-Pd2 in measuring Pd(II) in biological fluid.
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Figure 6. The confocol images of HeLa cells incubated with Umb-Pd2 in different conditions.
The cells were initially incubated with Umb-Pd2 (10 μM) for 30 min at 37 ℃, then treated with
(a-c) PBS buffer, (d-f) 100 μM Pd(0), (g-i) 50 μM Pd(II) or (j-l) 100 μM Pd(II) for another 30 min
at 37 ℃. Blue channel: 440 - 490 nm; Scale bar: 50 μm.
4. Conclusion
In summary, an umbelliferone derivative Umb-Pd2 as a small, steady, safe and selective
sensor for detecting Pd(II) was provided. It presented a ratiometric fluorescence change from
380 nm to 450 nm when triggered by Pd(II). Compared with the recent researches (Table S2, SI),
Umb-Pd2 indicated potential for applications with the linear range of 0 - 1.8 equivalent, the LOD
of 1.1 nM and the pH window of 5.0 - 10.0. The most attractive point was its selectivity towards
Pd(II) from Pd(0) convinced by both independent and coexistence systems. Among the
previously reports, most probes for Pd did not provide practical selectivity data. Some
examples raised and discussed the selectivity for Pd(0) [22,25,26,28-30,35,51], but very few
ones mentioned selectivity for Pd(II) [50]. Such a distinguishing ability of Umb-Pd2 was
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even available in biological imaging in living cells with a designed turn-on phenomena by
ultilizing the boundary between ultraviolet and visible light. This key point raised this work as a
rare and important example among all the published papers on palladium sensing. Thus,
Umb-Pd2 offered useful information for developing further tools on precise detection of Pd(II)
from Pd(0) in both biological and catalytic systems, being significant for daily chemistry and
public health.
Acknowledgments
This work is supported by the Public Science and Technology Research Funds Projects of
Ocean (No. 201505023), the National Undergraduate Innovation Program and the National
Science Foundation of China (No. J1210026).
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.saa.2019.XX.XXX.
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References
[1] S. Lee, G. Mair, Excelsior!: The Amazing Life of Stan Lee, New York, New York: Simon &
Schuster (2002) 160-160. ISBN 978-0684873053.
[2] G. P. Carman, N. Sun, Strain-mediated magnetoelectrics: Turning science fiction into reality,
MRS Bull. 43 (2018) 822-828.
[3] M. Jablonska, A. Krol, E. Kukulska-Zajac, K. Tarach, L. Chmielarz, K. Gora-Marek, Zeolite Y
modified with palladium as effective catalyst for selective catalytic oxidation of ammonia to
nitrogen, J. Catal. 316 (2014) 36-46.
[4] U. Bardi, S. Caporali, Precious Metals in Automotive Technology: An Unsolvable Depletion
Problem? Minerals 4 (2014) 388-398.
[5] F. Paul, J. Patt, J. F. Hartwig, Palladium-catalyzed formation of carbon-nitrogen bonds.
Reaction intermediates and catalyst improvements in the hetero cross-coupling of aryl halides and
tin amides, J. Am. Chem. Soc. 116 (1994) 5969-5970.
[6] R. F. Heck, J. P. Nolley, Palladium-catalyzed vinylic hydrogen substitution reactions with aryl,
benzyl, and styryl halides, J. Org. Chem. 37 (1972) 2320-2322.
[7] N. Miyaura, A. Suzuki, Palladium-catalyzed cross-coupling reactions of organoboron
compounds, Chem. Rev. 95 (1995) 2457-2483.
[8] K. Sonogashira, Development of Pd-Cu catalyzed cross-coupling of terminal acetylenes with
sp2-carbon halides, J. Organomet. Chem. 653 (2002) 46-49.
[9] I. Iavicoli, G. Carelli, A. Marinaccio, L. Fontana, E. J. Calabrese, Effects of sub-chronic
exposure to palladium (as potassium hexachloro-palladate) on cytokines in male Wistar rats, Hum.
Exp. Toxicol. 27 (2008) 493-497.
[10] K. Leopold, M. Maier, S. Weber, M. Schuster, Long-term study of palladium in road tunnel
dust and sewage sludge ash, Environ. Pollut. 156 (2008) 341-347.
[11] S. Zimmermann, C. Wolff, B. Sures, Toxicity of platinum, palladium and rhodium to Daphnia
magna in single and binary metal exposure experiments, Environ. Pollut. 224 (2017) 368-376.
[12] J. Kielhorn, C. Melber, D. Keller, I. Mangelsdorf, Palladium-a review of exposure and effects
to human health, Int. J. Hyg. Environ. Health 205 (2002) 417-432.
[13] C.D. Spicer, T. Triemer, B.G. Davis, Palladium-mediated cell-surface labeling, J. Am. Chem.
Soc. 134 (2011) 800-803.
12

ACCEPTED MANUSCRIPT
[14] D. Downey, Contact mucositis due to palladium, Contact Dermat. 21 (1989) 54-54.
[15] J. A. Marcusson, Contact allergies to nickel sulfate, gold sodium thiosulfate and palladium
chloride in patients claiming side-effects from dental alloy components, Contact Dermat. 34 (1996)
320-323.
[16] K. Benkhedda, B. Dimitrova, H. G. Infante, E. Ivanova, F. C. Adams, Simultaneous online
preconcentration and determination of Pt, Rh and Pd in urine, serum and road dust by flow
injection combined with inductively coupled plasma time-of-flight mass spectrometry, J. Anal. At.
Spectrom. 18 (2003) 1019-1025.
[17] B. Dimitrova, K. Benkhedda, E. Ivanova, F. C. Adams, Flow injection on-line
preconcentration of palladium by ion-pair adsorption in a knotted reactor coupled with
electrothermal atomic absorption spectrometry, J. Anal. At. Spectrom. 19 (2004) 1394-1396.
[18] C. Locatelli, D. Melucci, G. Torsi, Determination of platinum-group metals and lead in
vegetable environmental bio-monitors by voltammetric and spectroscopic techniques: critical
comparison, Anal. Bioanal. Chem. 382 (2005) 1567-1573.
[19] K. Van Meel, A. Smekens, M. Behets, P. Kazandjian, R. Van Grieken, Determination of
platinum, palladium, and rhodium in automotive catalysts using high-energy secondary target
X-ray fluorescence spectrometry, Anal. Chem. 79 (2007) 6383-6389.
[20] D. S. Risley, R. Chaddha, D. Mitchell, Retention, direct detection, and quantitation of
palladium (II) using high-performance liquid chromatography and evaporative light scattering
detection, J. Liq. Chromatogr. Relat. Technol. 40 (2017) 770-774.
[21] Heena, R. Kaur, S. Rani, A. K. Malik, A. Kabir, K. G. Furton, Determination of cobalt(II),
nickel(II) and palladium(II) ions via fabric phase sorptive extraction in combination with
high-performance liquid chromatography-UV detection, Sep. Sci. Technol. 52 (2017) 81-90.
[22] T. Yu, G. X. Yin, P. Yin, Y. Zeng, H. T. Li, Z.Y. Zhang, S.Z. Yao, A novel long-wavelength
fluorescent probe for discrimination of different palladium species based on Pd-catalyzed
reactions, RSC Adv. 7 (2017) 24822-24827.
[23] J. W. Yan, X. L. Wang, L. F. Zhou, L. Zhang, CV-APC, a colorimetric and red-emitting
fluorescent dual probe for the highly sensitive detection of palladium, RSC Adv. 7 (2017)
20369-20372.
[24] W. F. Luo, J. Li, W. S. Liu, A two-photon ratiometric ESIPT probe for fast detection and
13

ACCEPTED MANUSCRIPT
bioimaging of palladium species, Org. Biomol. Chem. 15 (2017) 5846-5850.
[25] T. Kuhana A, W. Y. Feng, G. Q. Feng, A simple but effective colorimetric and far-red to
near-infrared fluorescent probe for palladium and its application in living cells, Dyes Pigments
152 (2018) 112-117.
[26] W. F. Luo, W. S. Liu, A two-photon ratiometric ESIPT probe for the discrimination of
different palladium species and its application in bioimaging, J. Mater. Chem. B 4 (2016)
3911-3915.
[27] Y. C. Liu, K. Q. Xiang, M. Guo, B. Z. Tian, J. L. Zhang, A coumarin-based fluorescent probe
for the fast detection of Pd0 with low detection limit, Tetrahedron Lett. 57 (2016) 1451-1455.
[28] K. Wang, G. Q. Lai, Z. F. Li, M. M. Liu, Y. J. Shen, C. Y. Wang, A novel colorimetric and
fluorescent probe for the highly selective and sensitive detection of palladium based on Pd(0)
mediated reaction, Tetrahedron 71 (2015) 7874-7878.
[29] P. Wang, J. X. Xia, Y. Q. Gu, A novel NIR fluorescent probe for palladium detection based on
Pd(0) mediated reaction, Tetrahedron Lett. 56 (2015) 6491-6494.
[30] X. H. Wang, Z. Q. Guo, S. Q. Zhu, H. Tian, W. H. Zhu, A naked-eye and ratiometric
near-infrared probe for palladium via modulation of a π-conjugated system of cyanines, Chem.
Commun. 50 (2014) 13525-13528.
[31] K. Q. Xiang, Y. C. Liu, C. J. Li, B. Z. Tian, J. L. Zhang, A colorimetric and ratiometric
fluorescent probe for detection of palladium in the red light region, RSC Adv. 5 (2015)
52516-52521.
[32] L. Cui, W. P. Zhu, Y. F. Xu, X. H. Qian, A novel ratiometric sensor for the fast detection of
palladium specieswith large red-shift and high resolution both in aqueous solution andsolid state,
Anal. Chim. Acta 786 (2013) 139-145.
[33] F. L. Song, A. L. Gamer, K. Koide, A highly sensitive fluorescent sensor for palladium based
on the allylic oxidative insertion mechanism, J. Am. Chem. Soc. 129 (2007) 12354-12355.
[34] Y. Zhang, Y. X. Li, X. F. Yang, Y. M. Ding, Z. S. Zhao, X. L. Liu, Z. Yang, Y. Cui, A
single-state fluorescent with bright white-light emission in the solid station and
aggregation-induced emission enhancement compound for Pd0 detection, Talanta 179 (2018)
177-185.
[35] Y. B. Ding, S. Zhao, Q. Q. Wang, X. Yu, W. H. Zhang, Construction of a coumarin based
14

ACCEPTED MANUSCRIPT
fluorescent sensing platform for palladium and hydrazine detection, Sensor. Actuat. B-Chem. 256
(2018) 1107-1113.
[36] Z. W. Ma, X. Wang, Y. L. Sun, J. T. Liu, Y. Tong, Z. J. Liu, A novel fluorescent probe for the
highly selective and sensitive detection of palladium in aqueous medium, J. Fluoresc. 26 (2016)
1917-1921.
[37] J. Qin, X. M. Li, Z. Z. Chen, F. Feng, A highly selective and commercially available
fluorescence probe forpalladium detection, Sensor. Actuat. B-Chem. 232 (2016) 611-618.
[38] Z. Y. Xu, J. Li, S. Guan, L. Zhang, C. Z. Dong, Highly selective and sensitive fluorescence
chemosensor for the detection of palladium species based on Tsuji-Trost reaction, Spectrochim.
Acta A 148 (2015) 7-11.
[39] L. Y. Zhou, S. Q. Hu, H. F. Wang, H. Y. Sun, X. B. Zhang, A novel ratiometric two-photon
fluorescent probe for imaging of Pd2+ ions in living cells and tissues, Spectrochim. Acta A 166
(2016) 25-30.
[40] Y. Chen, B. Chen, Y. F. Han, A novel rhodamine-based fluorescent probe for the fluorogenic
andchromogenic detection of Pd2+ ions and its application in live-cellimaging, Sensor. Actuat.
B-Chem. 237 (2016) 1-7.
[41] Y. Long, Y. J. Bai, J. Zhou, B. Q. Yang, Selective and sensitive off-on probes specific for
palladium detection and their application in biological environments, J. Photoch. Photobio. A 332
(2017) 422-431.
[42] M. Z. K. Baig, S. Pawar, R. N. P. Tulichala, A. Nag, M. Chakravarty, A single fluorescent
probe as systematic sensor for multiple metal ions: Focus on detection and bio-imaging of Pd2+,
Sensor. Actuat. B-Chem. 243 (2017) 226-233.
[43] F. J. Huo, Y. Q. Zhang, J. Kang, J. B. Chao, Y. B. Zhang, C. X. Yin, A novel alkyne
compound as a Pd(II) fluorescent probe in aqueousmedium and its bioimaging, Sensor. Actuat.
B-Chem. 243 (2017) 429-434.
[44] A. Y. Mironenko, M.V. Tutov, A. A. Sergeev, Voznesenskiy, S. Y. Bratskaya, On/off
rhodamine based fluorescent probe for detection of Au and Pd in aqueous solutions, Sensor. Actuat.
B-Chem. 246 (2017) 380-394.
[45] A. Higashi, N. Kishikawa, K. Ohyama, N. Kuroda, A simple and highly selective fluorescent
sensor for palladium based on benzofuran-2-boronic acid, Tetrahedron Lett. 58 (2017) 2774-2778.
15

ACCEPTED MANUSCRIPT
[46] M. P. Yang, B. Q. Yang, L. L. Kang, L. Wang, L. F. Ma, J. Li, Y. Zhang, Two highly selective
fluorescence probes for imaging Pd2+ in cells and mice, Tetrahedron Lett. 58 (2017) 4769-4775.
[47] X. Z. Chen, H. M. Wang, X. D. Ma, M. Wang, Y. Y. Zhang, G. Gao, J. J. Liu, S. C. Hou,
Colorimetric and fluorescent probe for real-time detection of palladium (II) ion in aqueous
medium and live cell imaging, Dyes Pigments 148 (2018) 286-291.
[48] L. C. Li, R. F. Guan, M. M. Guo, P. Ning, R. Shao, X. M. Meng, A FRET based two-photon
fluorescent probe for ratiometric detectionof Pd2+ in living cells and in vivo, Sensor. Actuat.
B-Chem. 254 (2018) 949-955.
[49] J. Wen, Y. H. Lv, P. Y. Xia, F. Y. Liu, Y. Q. Xu, H. J. Li, S. S. Chen, S. G. Sun, A water-soluble
near-infrared fluorescent probe for specific Pd2+ detection, Bioorg. Med. Chem. 26 (2018)
931-937.
[50] J. L. Zhou, S. Xu, X. C. Dong, Z. J. Chen, W. L. Zhao, Near-infrared off-on fluorescent probe
for fast and selective detection of palladium (II) in living cells, J. Photoch. Photobio. A 355 (2018)
158-164.
[51] Q. F. Xia, S. M. Feng, D. D. Liu, G. Q. Feng, A highly selective and sensitive colorimetric
and near-infrared fluorescent turn-on probe for rapid detection of palladium in drugs and living
cells, Sensor. Actuat. B-Chem. 258 (2018) 98-104.
[52] B. L. Huo, M. Du, A. J. Gong, M. W. Li, L. Q. Fang, A. Shen, Y. R. Lai, X. Bai, Y. X. Yang, A
novel intramolecular cyclization-induced fluorescent "turn-on" probe for detection of Pd2+ based
on the Tsuji-Trost reaction, Anal. Methods 10 (2018) 3475-3480.
[53] H. L. Chen, X. X. Jin, W. X. Zhang, H. F. Lu, W. Shen, A new rhodamine B-based 'off-on'
colorimetric chemosensor for Pd2+ and its imaging in living cells, Inorg. Chim. Acta 482 (2018)
122-129.
[54] J. L. Zhang, L. Zhang, Y. M. Zhou, T. S. Ma, J. Y. Niu, A highly selective fluorescent probe
for the detection of palladium(II) ion in cells and aqueous media, Microchim. Acta 180 (2013)
211-217.
[55] Q. Huang, Y. M. Zhou, Q. Y. Zhang, E. Z. Wang, Y. H. Min, H. Qiao, J. L. Zhang, T. S. Ma, A
new “off-on” fluorescent probe for Pd2+ in aqueous solution and live-cell based on spirolactam
ring-opening reaction, Sensor. Actuat. B-Chem. 208 (2015) 22-29.
[56] Y. M. Zhou, J. L. Zhang, H. Zhou, Q. Y. Zhang, T. S. Ma, J. Y. Niu, A highly sensitive and
16

ACCEPTED MANUSCRIPT
selective “off-on” chemosensor for the visual detection of Pd2+ in aqueous media, Sensor. Actuat.
B-Chem. 171-172 (2012) 508-514.
[57] Y. M. Zhou, Q. Huang, Q. Y. Zhang, Y. H. Min, E. Z. Wang, A simple-structured acridine
derivative as a fluorescent enhancement chemosensor for the detection of Pd2+ in aqueous media,
Spectrochim. Acta A 137 (2015) 33-38.
[58] V. Sharma, A. K. Saini, S. M. Mobin, Multicolour fluorescent carbon nanoparticle probes for
live cell imaging and dual palladium and mercury sensors, J. Mater. Chem. B 4 (2016) 2466-2476.
[59] Y. P. Feng, A. Blanquer, J. Fornell, H. Y. Zhang, P. Solsona, M. D. Baro, S. Surinach, E.
Ibanez, E. Garcia-Lecina, X. Q. Wei, R. Li, L. Barrios, E. Pellicer, C. Nogues, J. Sort, Novel
Fe-Mn-Si-Pd alloys: insights into mechanical, magnetic, corrosion resistance and biocompatibility
performances, J. Mater. Chem. B 4 (2016) 6402-6412.
[60] A. Schrofel, G. Kratosova, I. Safarik, M. Safarikova, I. Raska, L.M. Shor, Applications of
biosynthesized metallic nanoparticles - A review, Acta Biomater. 10 (2014) 4023-4042.
[61] L. Sero, L. Sanguinet, S. Derbre, F. Boury, G. Brotons, S. Dabos-Seignon, P. Richomme, D.
Seraphin, Fluorescent self-assembled mono layers of umbelliferone: A relationship between
contact angle and fluorescence, Langmuir, 29 (2013) 10423-10431.
[62] A. C. Sedgwick, A. Hayden, B. Hill, S. D. Bull, R. B. P. Elmes, T. D. James, A simple
umbelliferone based fluorescent probe for the detection of nitroreductase, Front. Chem. Sci. Eng.
12 (2018) 311-314.
[63] M. O. Germoush, S. I. Othman, M. A. Al-Qaraawi, H.M. Al-Harbi, O. E. Hussein, G.
Al-Basher, M. F. Alotaibi, H. A. Elgebaly, M. A. Sandhu, A. A. Allam, A.M. Mahmoud,
Umbelliferone prevents oxidative stress, inflammation and hematological alterations, and
modulates glutamate-nitric oxide-cGMP signaling in hyperammonemic rats, Biomed.
Pharmacother. 102 (2018) 392-402.
[64] X. H. Jin, Y. Xu, X. H. Yang, X. L. Chen, M. H. Wu, J. G. Guan, L. S. Feng, Design, synthesis
and in vitro anti-microbial evaluation of ethylene/propylene-1H-1,2,3-triazole-4-methylene
-tethered isatin-coumarin hybrids, Curr. Top. Med. Chem. 17 (2017) 3213-3218.
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Figures Captions
Figure 1. Shematic illustration of selective detection for Pd(II) by Umb-Pd2.
Figure 2. General synthesis route of Umb-Pd2.
Figure 3. (a) The fluorescence emission spectra of Umb-Pd2 upon increasing concentrations
of Pd(II) (0 - 20 equiv.); (b) The logistic fitting in the range of 0 - 20 equivalent; (b-Inner) The
linear correlation in the range of 0 - 1.8 equivalent.
Figure 4. The intensity ratio (I450/I380) values indicated the selectivity of Umb-Pd2 towards
Pd(II) (a) from other analytes and (b) in the coexistence systems.
Figure 5. (a) The Pd(II)-induced depropargylation reaction of Umb-Pd2, (b) the 1H NMR data
and (c) the HRMS data comparison of corresponding compounds.
Figure 6. The confocol images of HeLa cells incubated with Umb-Pd2 in different conditions.
The cells were initially incubated with Umb-Pd2 (10 μM) for 30 min at 37 ℃, then treated
with (a-c) PBS buffer, (d-f) 100 μM Pd(0), (g-i) 50 μM Pd(II) or (j-l) 100 μM Pd(II) for
another 30 min at 37 ℃. Blue channel: 440 - 490 nm; Scale bar: 50 μm.
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An umbelliferone-derivated fluorescent sensor for selective
detection of palladium(II) from palladium(0) in living cells
Xu-Ping Zhang‡, Qing Yuan‡, Ya-Lin Qi, Da-Jun Zheng, Qi-Xing Liu, Bao-Zhong Wang*, Yu-Shun
Yang* and Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing, 210023, China.
*Corresponding authors. Tel. & Fax: +86-25-89682572; E-mail: zhuhl@nju.edu.cn;
ys_yang@nju.edu.cn; wbz@nju.edu.cn.
‡Both authors contributed equally to the work
For quantitative and selective detection of palladium, an umbelliferone
derivative Umb-Pd2 was provided as a small, steady, safe and selective sensor for
detecting Pd(II). It indicated advantages including sensitive, wide pH tolerance,
applicable linear range and low toxicity. The most attractive point was its explicit
selectivity towards Pd(II) from Pd(0) in both independent and coexistence systems,
being different from usual investigations mainly claiming selectivity for Pd(0). This
distinguishing ability was further utilized in imaging in living cells. Umb-Pd2 supplied
a potential approach for further improvement and applications in both daily
chemistry and public health.
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Graphical abstract
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Highlights
> Explicit selectivity towards Pd(II) from Pd(0) in both independent and coexistence
systems
> Low detection limit, wide pH tolerance, applicable linear range and low toxicity
> Applied in biological imaging with a designed turn-on phenomena and also in fluid
> Simple regeneration for economic and environmental consideration
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