A new hydroxynaphthyl benzothiazole derived fluorescent probe for highly selective and sensitive Cu2?+ detection

Article abstract of DOI:10.1016/j.saa.2016.06.045

A new reactive probe, 1-(benzo[d]thiazol-2-yl)naphthalen-2-yl-picolinate (BTNP), was designed and synthesized. BTNP acts as a highly selective probe to Cu^2?+ in DMSO/H₂O (7/3, v/v, Tris-HCl 10?mM, pH?=?7.4) solution based on Cu^2?+ catalyzed hydrolysis of the picolinate ester moiety in BTNP, which leads to the formation of an ESIPT active product with dual wavelength emission enhancement. The probe also possesses the advantages of simple synthesis, rapid response and high sensitivity. The pseudo-first-order reaction rate constant was calculated to be 0.205?min^??1. Moreover, application of BTNP to Cu^2?+ detection in living cells and real water samples was also explored.

Full text of DOI:10.1016/j.saa.2016.06.045

A new hydroxynaphthyl benzothiazole derived fluorescent probe for highly
selective and sensitive Cu2 + detection
Lijun Tang, Ping He, Keli Zhong, Shuhua Hou, Yanjiang Bian
PII:
DOI:
Reference:
S1386-1425(16)30374-2
doi: 10.1016/j.saa.2016.06.045
SAA 14522
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Revised date:
Accepted date:
9 April 2016
22 June 2016
28 June 2016
Please cite this article as: Lijun Tang, Ping He, Keli Zhong, Shuhua Hou, Yanjiang Bian,
A new hydroxynaphthyl benzothiazole derived fluorescent probe for highly selective and
sensitive Cu2 + detection, (2016), doi: 10.1016/j.saa.2016.06.045
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ACCEPTED MANUSCRIPT
A new hydroxynaphthyl benzothiazole derived fluorescent probe for highly
selective and sensitive Cu2+ detection
Lijun Tang, Ping He, Keli Zhong, Shuhua Hou, Yanjiang Bian
Department of Chemistry, Bohai University, Jinzhou 121013, China
Abstract: A new reactive probe, 1-(benzo[d]thiazol-2-yl)naphthalen-2-yl-picolinate
(BTNP), was designed and synthesized. BTNP acts as a highly selective probe to
Cu2+ in DMSO/H2O (7/3, v/v, Tris-HCl 10 mM, pH =7.4) solution based on Cu2+
catalyzed hydrolysis of the picolinate ester moiety in BTNP, which leads to the
formation of an ESIPT active product with dual wavelength emission enhancement.
The probe also possesses the advantages of simple synthesis, rapid response and high
sensitivity. The pseudo-first-order reaction rate constant was calculated to be 0.205
min-1. Moreover, application of BTNP to Cu2+ detection in living cells and real water
samples were also explored.
Keywords: Cu2+ recognition, Cu2+ catalyzed hydrolysis, Benzothiazole, ESIPT, cell
imaging
1. Introduction
Transition and heavy metal ions play crucial roles in many biological and
environmental processes [1]. However, they can be toxic at high concentrations and
thus have extremely impact on the environment as well as on human health [2-5]. In
particular, Cu2+, after iron and zinc, is the third most abundant essential transition
metal ion in the human body, and plays pivotal roles in several natural processes [6].
 Corresponding author. Tel.: +86-416-3400302; E-mail address: ljtang@bhu.edu.cn
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It is also known to be a catalytic cofactor for many metalloenzymes and proteins in
organisms [7-9]. Abnormal level of Cu2+ ions in the human body is closely associated
with many diseases, such as myelopathy, neutropenia, Alzheimer's disease, Menkes
syndrome, Amyotrophic lateral sclerosis, and Wilson’s disease [10-15]. Therefore, the
efficient and convenient methods for monitoring the existence of Cu2+ in biological
samples or environment are of importance. Several analytical methods have been
developed to detect Cu2+, including inductively coupled plasma mass spectrometry
(ICP–MS) [16], inductively coupled plasma atomic emission spectrometry (ICP–AES)
[17] and atomic absorption spectrometry (AAS) [18], etc. However, most of these
techniques require complicated sample pretreatment, expensive and sophisticated
instrumentation. In contrast to these methods, fluorescence spectrometry has been
proved to be an ideal tool for trace amount metal ion detection due to its high
sensitivity, operational simplicity and real-time monitoring properties.
Recently, many fluorescent probes for Cu2+ detection have been developed based
on different strategies such as coordination to receptors [19-23], Cu2+-mediated
hydrolysis of amides and esters [24-29], oxidation of phenothiazine, phenol and
dihydrorosamine [30-33], etc. However, many of them still have some limitations such
as suffering from incomplete selectivity over the competing metal ions including Fe3+,
Co2+, Ni2+ and Hg2+ due to the similar ionic radius and chemical properties [34-36].
Thus, it is still highly desirable to develop new simple-structured fluorescent probes
to monitor the existence of Cu2+ ions in environmental and biological systems.
Recently, fluorescent dyes with excited-state intramolecular proton transfer (ESIPT)
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characteristic have attracted great attention in probe design due to their intriguing
photophysical properties including dual wavelength emission and large Stokes shift
[37-39]. 2-(2′-hydroxyphenyl)benzothiazole (HBT), as a typical ESIPT active
fluorophore, has drawn considerable attention, and many HBT derived fluorescent
probes based on modulating ESIPT have been extensively explored [40-48]. However,
fluorescent
probes
exploiting
HBT
analogue
such
as
1-(benzo[d]thiazol-2-yl)naphthalen-2-ol (1), a well-known ESIPT active compound
[49], as the signaling fluorophore is still rare [50, 51]. Recently, some fluorescent
probes based on Cu2+ catalyzed hydrolysis of picolinoyl-ester have been documented
[52, 53]. For example, Han et al reported a HBT-based fluorescent probe CS1
(Scheme 1) [54], which displays fluorescence ‘turn on’ response to Cu2+ through Cu2+
catalyzed hydrolysis of the picolinoyl-ester moiety of CS1. Inspired by this and other
successful works, we herein synthesized a new hydroxynaphthyl benzothiazole
derived compound BTNP (Scheme 1), which is hoped to function as a fluorescence
‘off-on’ probe for Cu2+ detection based on Cu2+ catalyzed hydrolysis. Moreover,
compound 1, the assumed BTNP hydrolysis product, contains a larger π-conjugation
framework than that of HBT, is anticipated to behave the desired ESIPT featured dual
wavelengths emission with emission bathochromic shift, which thus enables
ratiometric emission changes. Further studies reveal that probe BTNP indeed exhibits
high selectivity toward Cu2+ through dual wavelengths emission enhancement with a
ratiometric sensing behavior.
Scheme 1.
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2. Experimental section
2.1 Reagents and Apparatus
Unless otherwise noted, reagents and solvents were obtained from commercial
suppliers and used as received. 1-(Benzo[d]thiazol-2-yl)naphthalen-2-ol (1) was
13
prepared according to the reported method [51]. 1H NMR and C NMR spectra were
checked on an Agilent 400 MR spectrometer, and the chemical shifts (δ) were
expressed in ppm and coupling constants (J) in Hertz. High-resolution mass spectra
(HRMS) were measured using an LTQ Orbitrap XL hybrid mass spectrometer
(Thermo-Fisher Scientific, San Jose, CA, USA). Fluorescence measurements were
carried out on a Sanco 970-CRT spectrofluorometer (Shanghai, China). The pH
measurements were made with a Model PHS-25B meter (Shanghai, China). Cell
imaging was observed under an Olympus FV1000-IX81 confocal laser scanning
microscope with excitation at 405 nm.
2.2 Synthesis of probe BTNP
A mixture of compound 1 (277 mg, 1.0 mmol), dicyclohexyl carbodiimide (DCC)
(227 mg, 1.10 mmol), 4-dimethylaminopyridine (DMAP) (12 mg, 0.10 mmol) and
picolinic acid (124 mg, 1.01 mmol) in dry dichloromethane (20 mL) was stirred for 24
hours at room temperature. After addition 60 mL of water, the mixture was extracted
with dichloromethane (3×20 mL). The combined organic layers were dried and
filtered. The solvent was removed by rotary evaporation and the crude product was
purified by column chromatography (ethyl acetate/petroleum ether = 1:3, v/v) to give
o
1
BTNP as white power (271 mg, 71%). M.p. 107-109 C. H NMR (400 MHz,
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DMSO-d6) δ 8.72 (d, J = 4.4 Hz, 1H), 8.31 (d, J = 9.2 Hz, 1H), 8.14 (d, J = 8.0, 2H),
8.10 (d, J = 7.6 Hz, 2H), 8.01-7.97 (m, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.8
Hz, 1H), 7.69-7.61 (m, 3H), 7.58-7.55 (m, 1H), 7.51-7.47 (m, 1H). 13C NMR (100
MHz, DMSO-d6) δ 163.88, 161.24, 153.16, 150.60, 147.52, 146.49, 138.22, 135.91,
132.68, 132.12, 131.86, 128.90, 128.59, 127.03, 126.96, 126.24, 125.38, 123.65,
122.70, 122.53, 122.46. HRMS (ESI+): Calcd. for C23H15N2O2S ([M+H])+,
383.0854; Found, 383.0855.
2.3 Spectroscopic measurements
Stock solutions of metal ions (10.0 mM, used as chloride or nitrate salts) including
CuCl2, AgNO3, Al(NO3)3, CdCl2, CoCl2, CrCl3, FeCl3, HgCl2, KCl, MgCl2, NaCl,
NiCl2, Sr(NO3)2, Pb(NO3)2, ZnCl2, CaCl2, MnCl2, FeCl2, and BaCl2 were prepared in
deionised water. Stock solution of BTNP (1.0 mM) was prepared in DMSO, this stock
solution was further diluted with a mixed solution of DMSO/H2O (7/3, v/v, Tris-HCl
10 mM, pH =7.4) to 10 μM for optical measurement. For the sensitivity measurement,
different concentrations of Cu2+ ions were added to the assay solution, and the
fluorescence spectra were recorded. The selectivity was checked by addition the same
dose of Cd2+, Co2+, Hg2+, Ni2+, Ag+, Pb2+, Sr2+, Ba2+, Fe2+, Mn2+, Fe3+, Zn2+, Al3+, Cr3+,
Mg2+, K+, Ca2+, and Na+ into the assay solution.
2.4 Cell culture and fluorescence imaging experiments
MCF-7 (human breast carcinoma) cells were obtained from Institute of Basic
Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS). MCF-7
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
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supplemented with 10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and
95% air at 37 °C. Grow MCF-7 cells in the exponential phase of growth on 35-mm
glass-bottom culture dishes (Φ 20 mm) for 1-2 days to reach 70-90% confluence. The
culture medium was removed and the cells were washed three times with PBS buffer,
then the cells were incubated BTNP (10 μM) for 30 min at 37 °C, after washing three
times with PBS buffer, the cell imaging was conducted. The probe incubated cells
were subsequently incubated with CuCl2 (100 μM) for 30 min at 37 °C, washed three
times with PBS buffer and then cell imaging was collected.
3. Results and Discussion
Probe BTNP was facilely synthesized by condensation of compound 1 with
picolinic acid in the presence of DCC (Scheme 1), and the structure of BTNP was
1
13
fully characterized by H NMR, C NMR and HRMS analysis (see Figs. S1-S3 for
detailed spectrum). After screening the working solvent media, a cosolvent of
DMSO/H2O (7/3, v/v, Tris-HCl 10 mM, pH =7.4) was selected for fluorescence
spectra measurement. Subsequently, the fluorescence response of BTNP toward
different metal ions was assayed (Fig. 1). On addition of Cu2+ to BTNP solution, two
dramatically enhanced fluorescence emission bands at 428 nm and 475 nm were
observed, which could be attributed to the Cu2+ catalyzed hydrolysis of BTNP that
resulting in formation of the ESIPT active 1. Compared with Han’s probe CS1 that
emits at 460 nm on treatment with Cu2+ [54], Cu2+ treated BTNP exhibits a
bathochromic shifted emission at 475 nm, which is attributed to the larger
π-conjugation framework of 1 than that of HBT. In additon, the anticipated dual
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wavelengths emission indeed occurred. These features make BTNP is distinguishable
from the structural similar HBT derived probe CS1. In addition, the Cu2+ induced
appearance of cyanine blue emission color of BTNP solution was observable under
illumination with a portable UV lamp at 365 nm (Fig. 1, inset). However, individual
addition of other metal ions, including Cd2+, Co2+, Hg2+, Ni2+, Ag+, Pb2+, Sr2+, Ba2+,
Fe2+, Mn2+, Fe3+, Zn2+, Al3+, Cr3+, Mg2+, K+, Ca2+, and Na+, caused no distinct
fluorescence enhancement at 428 nm and 475 nm. Therefore, an excellent selectivity
of BTNP toward Cu2+ was achieved.
Fig. 1.
To further confirm the Cu2+ promoted hydrolysis event, reaction of probe BTNP
with Cu2+ in DMSO-H2O (7:3, v/v) was conducted separately. Thin layer
chromatography (TLC) monitoring of the reaction process show that a compound
with a similar Rf value of 1 was obtained (Fig. S4). The isolated product was then
1
subjected to H NMR spectrum comparison with that of compound 1, and they have
almost identical observed spectrum (Fig. 2), demonstrating the Cu2+-induced
hydrolysis of BTNP to give 1. Coupled with the observed dual wavelength emission
of 1, the Cu2+ detection mechanism was illustrated in Scheme 2.
Fig. 2.
Scheme 2.
The Cu2+ promoted hydrolysis kinetics of BTNP was further investigated via
mornitoring the time-dependent fluorescence changes of BTNP solution in the
presence of 1.0 equiv. of Cu2+. The results show that the fluorescence intensity of
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BTNP solution increases with prolonging the stocking time and reaches a plateau
after 0.5 h of Cu2+ addition (Fig. S5). Thus, in the following investigations, the
fluorescence spectra were checked after 0.5 h of Cu2+ addition. The reaction kinetics
of BTNP in the presence of 1.0 equiv of Cu2+ according to a pseudo-first-kinetic was
then investigated (Fig. 3) with the equation It = Imax+A×exp(-k×t)[55-57]. In the
equation, It represents the fluorescence intensity (475 nm) of the test solution at time
t, Imax represents the maximum fluorescence intensity (475 nm) of the test solution.
The pseudo-first-order rate constant k was calculated to be 0.205 min-1, indicative of
the remarkable high sensitivity of BTNP for Cu2+ detection.
Fig. 3.
Subsequently, the fluorescence titration experiment was conducted to evaluate the
sensing behavior of BTNP to Cu2+. As shown in Fig. 4, upon successive addtion of
Cu2+ (0 to 1.0 equiv.) to BTNP solution, the emission intensities of the solution at 428
nm and 475 nm increased gradually, which was attributed to the generation of ESIPT
active compound 1 by Cu2+ catalyzed hydrolysis of the ester bond in BTNP. In
addition, the emission intensity ratio of F475/F428 showed a good linearity within Cu2+
concentration ranges from 0 to 2 μM (Fig. 4, inset). The observed ratiometric sensing
behavior of BTNP towards Cu2+ is superior to the single emission intensity-dependent
performance of reported probe CS1 [54], since the ratiometric emission output can
supply built-in correction property and thus can overcome the environmental and
probe concentration effects. From the titration profile, the association constant (Ka) of
BTNP and Cu2+ was estimated to be 5.72×106 M-1 by non-linear least squares fitting
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of the titration data based on a 1:1 binding equation (R2 = 0.9958) (Fig. S6).
Furthermore, the detection limit of BTNP to Cu2+ was then calculated. Plotting the
normalized fluorescence intensity against Log[Cu2+] provides a nice linear
relationship (Fig. 5), and the point of this line crossed the ordinate axis is regarded as
the detection limit [58-60], which was found to be 1.34×10-6 M. It was much lower
than the limit of Cu2+ concentration (20 μM) in drinking water permitted by the U.S.
Environmental Protection Agency.
Fig. 4. and Fig. 5.
For a good sensing probe, the anti-interference ability is another important
requirement. Thus, the influences of other potential competitive metal ions on Cu2+
detection were assayed. As depicted in Fig. 6, except for Cu2+, other potential
competitive metal ions did not promote noticeable fluorescence intensity changes of
probe BTNP solution, demonstrating that the Cu2+ recognition process has a good
anti-interference ability in the presence of other metal ions. This implies that probe
BTNP can be used in environmental and biological systems for Cu2+ detection even
in the presence of other competitive metal ions.
Fig. 6.
To further evaluate the practicability of BTNP for Cu2+ detection, influence of pH
on fluorescence changes of BTNP (at 475 nm) with and without Cu2+ was also
investigated (Fig. 7). BTNP solution in the presence and absence of Cu2+ exhibits
nonfluorescence within pH range from 2 to 5, which may attributed to the protonation
of pyridine nitrogen atom in BTNP and thus make it lost its binding ability with Cu2+.
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However, BTNP itself displays increased fluorescence emission under higher pH
(8-10) conditions, which is attributed to the hydroxide ion induced hydrolysis of
BTNP. The Cu2+ induced fluorescence enhancement of BTNP was observed within
the pH range from 6 to 8, suggesting that BTNP could be used for Cu2+ detection at
near neutral conditions.
Fig. 7.
To corroborate the potential utility of probe BTNP for biological applications, we
performed the live-cell imaging experiments. MCF-7 cells were incubated with
o
BTNP (10 μM) for 30 min at 37 C exhibiting no observable fluorescence iamge
collected form the green channel (425 nm to 525 nm) (Fig. 8A). Subsequently, the
BTNP incubated MCF-7 cells were washed three times with PBS buffer, and futher
o
incubated with CuCl2 (100 μM) for another 30 min at 37 C, obvious intracellular
green fluorescence can be observed (Fig. 8D). These results indicate that probe BTNP
is cell permeable and capable of detect Cu2+ in living cells.
Fig. 8.
The potential utilities of BTNP were also checked by ‘proof-of-concept’
experiments [61,62]. Tap, lake and river water were collected from laboratory, the
pool of Tinglin lake in Bohai University, and Xiaoling river (Linghe district, Jinzhou),
respectively. Cu2+ (0 to 0.6 equiv.) were spiked into these samples before BTNP (10
μM) was added. The results show that the fluorescence intensities were proportional
to the spiked Cu2+ concentration from 0 to 6 μM (lower than the limit of Cu2+ (∼15.7
μM)) in drinking water defined by the Standardization Administration of the People’s
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Republic of China) in all the three natural water samples (Fig. 9). Therefore, the
presented probe BTNP has potential applicability for trace Cu2+ detection in these
three complicated environmental systems.
Fig. 9.
4. Conclusion
In summary, we have synthesized a simple ESIPT-based fluorescence probe (BTNP)
for Cu2+ recognition based on Cu2+ catalyzed hydrolysis reaction. Probe BTNP
displays rapid response, highly selective, sensitive and ratiometric recognition to Cu2+
with excellent anti-interference ability. Application of BTNP to detect Cu2+ in living
cells and real water samples were also conducted, and the results indicate that BTNP
posesses potential applicability for Cu2+ detection in environmental and biological
samples.
Acknowledgments
The project was supported by the National Natural Science Foundation of China
(Nos. 21476029, 21176029) and the Program for Liaoning Excellent Talents in
University (No. LR2015001).
Supplementary data
Supplementary data (Figures S1-S6) associated with this article can be found in the
online version.
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Figures and Schemes
Scheme 1 Synthesis of probe BTNP, and the structure of reported CS1.
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Scheme 2. The sensing mechanism of BTNP for Cu2+.
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Fig. 1. Fluorescence spectra changes of BTNP (10 μM) in DMSO/H2O (7/3, v/v,
Tris-HCl 10mM, pH =7.4) solution upon respective addition 1.0 equiv. of different
metal ions (λex = 365 nm).
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Fig. 2. 1H NMR spectra(DMSO-d6) comparison of probe BTNP (a), compound 1 (b)
and the product obtained after reaction of the probe BTNP with CuCl2 (c).
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Fig. 3. The kinetic study of the response of BTNP (10 μM) to Cu2+(1.0 equiv) under
pseudo-first-order conditions in DMSO/H2O (7/3, v/v, Tris-HCl 10mM, pH =7.4)
solution.
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Fig. 4. Fluorescence spectra changes of BTNP (10 μM) in DMSO/H2O (7/3, v/v,
Tris-HCl 10mM, pH =7.4) solution upon incremental addition of Cu2+ (0 to 1.0
equiv.). λex = 365 nm.
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Fig. 5. Normalized fluorescence intensity (I-Imin)/(Imax-Imin) of BTNP (10 μM) as a
function of Log[Cu2+] in DMSO/H2O (7/3, v/v, Tris-HCl 10mM, pH =7.4) solution.
λex = 365 nm, λem = 475 nm.
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Fig. 6. Fluorescence intensity (475 nm) changes of BTNP (10 μM) in DMSO/H2O
(7/3, v/v, Tris-HCl 10mM, pH =7.4) solution upon indivisual addition of 1.0 equiv. of
miscellaneous metal ions (gray bars) and followed by further addition of 1.0 equiv. of
Cu2+ (red bars). λex = 365 nm.
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Fig. 7. The fluorescence intensity changes (at 475 nm) of BTNP with and without 1.0
equiv. of Cu2+ in DMSO/H2O (7/3, v/v, Tris-HCl 10mM, pH =7.4) solution at various
pH conditions. λex = 365 nm.
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Fig. 8. Fluorescence images of MCF-7 cells incubated with probe BTNP (10 μM) for 30
o
min at 37 C (A, from the dark field; B, from the bright field; C, merged image of A
o
and B) and further incubated with 100 μM of Cu2+ for 30 min at 37 C (D, from the
dark field; E, from the bright field; F, merged image of D and E).
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Fig. 9. Linear fluorescence intensities of BTNP (10 μM) upon addition of Cu2+ (0-6
μM) in three real water samples.
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Graphical abstract
A new fluorescent probe BTNP that exhibits highly selective recognition to Cu2+ in
DMSO/H2O (7/3, v/v, Tris-HCl 10 mM, pH =7.4) solution has been developed.
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Highlights
1) A new hydroxynaphthyl benzothiazole derived fluorescent probe BTNP has been
developed.
2) BTNP exhibits high selectivity to Cu2+ through Cu2+ catalyzed hydrolysis.
3) Cu2+ detection by BTNP displays two wavelength enhancements with ratiometric
behavior.
4) BTNP is applicable to Cu2+ detection in living cells and real water samples.
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