Cu2+-selective fluorescent chemosensor based on coumarin and its application in bioimaging

Article abstract of DOI:10.1039/c1dt11123j

A new fluorescent sensor L1 based on coumarin was synthesized. It shows high sensitivity and selectivity toward Cu²⁺ in aqueous solution. The complexation mode and corresponding quenching mechanism were elucidated by ESI-MS and DFT calculations

Full text of DOI:10.1039/c1dt11123j

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COMMUNICATION
Cu2+-selective fluorescent chemosensor based on coumarin and its application
in bioimaging†
Liang Huang,a Ju Cheng,b Kefeng Xie,c Pinxian Xi,a Fengping Hou,a Zhengpeng Li,a Guoqiang Xie,a
Yanjun Shi,a Hongyan Liu,a Decheng Baib and Zhengzhi Zeng*a
Received 15th June 2011, Accepted 18th August 2011
DOI: 10.1039/c1dt11123j
A new fluorescent sensor L1 based on coumarin was syn-
thesized. It shows high sensitivity and selectivity toward
Cu2+ in aqueous solution. The complexation mode and
corresponding quenching mechanism were elucidated by ESI-
MS and DFT calculations. In addition, biological imaging
studies have demonstrated that L1 can detect Cu2+ in living
cells.
been used with some success in biological applications.6 However,
some of them are not qualified for practical application due
to shortcomings such as slow response, low water solubility,
cross-sensitivities toward other metal cations and cytotoxicities
of the ligand. Therefore, the development of a highly sensitive
and selective fluorescent chemosensor for the detection of Cu2+
with a fast time response in aqueous solution is necessary. In
this communication, a highly sensitive and selective fluorescent
chemosensor based on coumarin for Cu2+ in aqueous solution is
demonstrated. An imaging study of Cu2+ in living cells is also
successfully shown.
Coumarin and its derivatives are excellent chromogenic and
fluorogenic dyes that are widely utilized as reporters in chemosen-
sors. In this communication, we report new coumarin-derived L1
bearing a benzoylhydrazine unit, which is demonstrated as an
on–off chemosensor for Cu2+ in aqueous media. To evaluate the
influence of hydrazide on the fluorescent system of coumarin,
the similar molecule L2 was accordingly synthesized (Fig. 1).
The synthetic pathways to fluorogenic molecules L1 and L2 are
summarized in Scheme S1 (ESI†). Compounds L1 and L2 were
synthesized in 82 and 76% yields, respectively. The structures of
L1 and L2 were confirmed by 1H NMR, 13C NMR, and FAB-MS
(Fig. S16–S20 ESI†). The structure of L2 was further confirmed
by X-ray crystallographic analysis (Fig. 3).
Chemosensors, small abiotic molecules that signal the presence
of analytes, typically combine two components: a recognition site
that binds the target substrate and a readout system that signals
binding.1 Of the many different kinds of optical sensors, fluores-
cent chemosensors have several advantages over the other methods
due to their intrinsic sensitivity, straightforward application to
fiber optical-based detection, and real-time monitoring with fast
response time.2 Recently, the development of selective and sensitive
imaging tools capable of rapidly monitoring heavy and transition
metal ions (HTM), such as Hg2+, Zn2+, and Cu2+ ions has attracted
considerable attention due to the environmental and biological
relevance of such metal ions.3–6 In this regard, sensors directed
toward the detection of divalent copper have enjoyed particular
attention. Copper is third in abundance (after Fe2+ and Zn2+)
among the essential heavy metal ions in the human body and plays
a pivotal role in a variety of fundamental physiological processes
in organisms ranging from bacteria to mammals.7 However, if
unregulated, copper can cause oxidative stress and disorders
associated with neurodegenerative diseases, such as Alzheimer’s
disease, Wilson’s disease, and Menke’s disease.8 Owing to the
Janus-faced properties of copper in organisms, many fluorescent
probes for Cu2+-selective detection have been reported and have
aKey Laboratory of Nonferrous Metals Chemistry and Resources Utilization
of Gansu Province, State Key Laboratory of Applied Organic Chemistry
and College of Chemistry and Chemical Engineering, Lanzhou University,
Lanzhou, 730000, PR China. E-mail: zengzhzh@yahoo.com.cn; Fax: +86
931 8912582; Tel: +86 931 8610877
Fig. 1 Chemical structures of L1 and L2.
The absorption spectrum of L1 (5 mM) in H2O/DMSO (9 : 1,
v/v) exhibited a strong band at 451 nm (Fig. S1 ESI†). Upon
addition of Cu2+ (0–2 equiv.), the absorption maximum at 451 nm
gradually decreased while the absorption at 490 nm increased. This
result indicated that the hydrazide was involved in coordination
with Cu2+. In the presence of other metal ions, the absorption
spectra did not show significant change under identical conditions.
The fluorescence spectrum of L1 in the same medium was
bSchool of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000,
PR China
cLanzhou Petrochemical Research Center, Petrochemical Research Institute,
PetroChina, Lanzhou, 730060, PR China
† Electronic supplementary information (ESI) available: Instruments and
experimental procedures; synthesis routes and characteristic data; supple-
mentary spectra; NMR spectra. CCDC reference numbers 829291 and
829292. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1dt11123j
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characterized by a strong emission of the coumarin moiety at
523 nm. Fluorescent titration experiments of L1 in the presence
and absence of Cu2+ were carried out in different pH values. The
results showed that L1 exhibited high sensitivity and selectivity for
Cu2+ within the pH range of about 5 to 8 (Fig. S3 ESI†).
and dichloromethane (Fig. 3). According to the structure of the
complex of L2-Cu2+, we can deduce a possible coordination mode
of L1-Cu2+. As shown in Scheme 1, the sensors are the most likely
to chelate with metal ions via their carbonyl O, imino N and
coumarin O atoms.
The complexation of Cu2+ with L1 was investigated by means
of a fluorescence titration in H2O/DMSO (9 : 1, v/v). L1 shows
very strong luminescence (U0 = 0.289) in H2O/DMSO (9 : 1, v/v)
solution. On addition and gradual increase of [Cu2+] (0–5 mM) to
the fluorescent solution of L1 (5 mM), the fluorescence intensity
was almost completely quenched (quenching efficiency at 523 nm,
(I0 - I)/I0 ¥ 100 = 98%) and the quantum yield was changed to
0.024 (Fig. 2). By measuring the maximum emission of L1-Cu2+,
the fluorescence change of L1 responded to the range of 0–10 mM
of [Cu2+]. We then found that L1 showed a 0.1 mM detection limit,
sufficient to sense the Cu2+ concentration in the blood system. The
association constant for Cu2+ was estimated to be 6.4 ¥105 M-1 in
the H2O/DMSO (9 : 1, v/v) solution on the basis of linear fitting
of the titration curve assuming a 1 : 1 stoichiometry (Fig. S4–6
ESI†). This binding mode was also supported by a Job plot.
Fig. 3 Crystal structure of L2 and L2-Cu2+. All hydrogen atoms are
omitted for clarity (50% probability level for the thermal ellipsoids).
Fig. 2 Fluorescence spectra of L1 (5 mM) in H2O/DMSO (9 : 1, v/v) upon
addition of Cu2+ (0–2 equiv.). Inset: Job plot according to the method for
continuous variations, indicating the 1 : 1 stoichiometry for L1-Cu2+ (the
total concentration of L1 and Cu2+ is 5 mM). (lex = 450 nm).
To improve the sensitivity and magnify the selectivity, the
response of the L1-Cu2+ complex toward physiologically and
environmentally important anions was investigated through flu-
orescence spectra. The results indicated that only S2- and P2O7
2-
Scheme 1 Probable complexation mechanism of L1 with Cu2+
.
can efficiently enhance the fluorescence intensity of the L1-Cu
-
-
system over other anions such as F-, Cl-, Br-, I-, ClO4 , NO3 ,CN-,
To understand the selectivity and the configuration of L1-Cu2+,
we carried out density functional theory (DFT) calculations with
B3LYP exchange functionals using the Gaussian 09 package.
The 6-31G basis sets were used except for Cu(NO3)2, where a
LANL2DZ effective core potential (ECP) was employed. The
optimized configuration is shown in Fig. 4, which shows that Cu2+
ions occupy the coordination centers of L1 and the whole molecule
forms a nearly planar structure. The Cu–N bond lengths are 2.00
-
HPO4- or HSO3 . Moreover, this sensing for L1-Cu2+ is also hardly
interfered with by commonly coexistent ions (Fig.S7–10 ESI†).
In order to understand the crucial role of the specific coordi-
nation environment of the L1-Cu2+ system, we also synthesized
L2 as a control. The fluorescence intensity of L2 was considerably
quenched in the presence of 1.0 equiv. of Cu2+ in H2O/DMSO
(9 : 1, v/v) solution. But the quantum yield of L2 (U = 0.062)
was much smaller compared to the quantum yield of L1 (U =
0.289) in the water/DMSO (9 : 1, v/v) solution due to the presence
of the OH oscillator in L2 (Fig. S10–11 ESI†). Luckily, we
obtained crystals of the complex L2-Cu2+ in a mixture of ethanol
˚
˚
A, and the Cu–O bond lengths are 2.03 A (Cu–coumarin) and 1.95
˚
A (Cu–benzoylhydrazine), respectively. Based on these data, it is
indicated that the design of the semirigid structure can effectively
create suitable space to better match corresponding ions.
10816 | Dalton Trans., 2011, 40, 10815–10817
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to be accumulated. Hep G2 (liver cancer) cells were incubated
with 5 mM L1 for 0.5 h at 37 ◦C and washed with PBS buffer
(pH 7.4) to remove the remaining L1, and then the treated cells
were incubated with Cu2+ (10 mM) in culture medium for 2 h at
37 ◦C. After incubation under the conditions, the cells were washed
with PBS buffer and imaged using a fluorescence microscope. Hep
G2 cells incubated with L1 initially display a strong fluorescence
image, but the fluorescence image became faint in the presence of
Cu2+ (Fig. 6a–c). Lowering the concentration of L1 to 1.0 mM, we
also can obtain the confocal image changes (Fig. S15 ESI†).
Fig. 4 Calculated energy-minimized structure of L1 with Cu(NO3)2.
An important feature of the chemosensor is its high selectivity
toward the analyte over the other competitive species. To validate
the selectivity of L1 in practice, some other metal ions including
alkali, alkaline earth, and transition metal ions were added into a
solution of L1 under the same conditions (H2O/DMSO 9 : 1, v/v).
As shown in Fig. 5, only Cu2+ elicited a prominent fluorescence
intensity decrease, while the other metal ions, such as K+, Ag+,
Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+ and
Zn2+ did not show any significant fluorescence change under the
identical conditions. In addition, the decreases of fluorescence
intensity resulting from the addition of the Cu2+ ion were not
influenced by the subsequentaddition of miscellaneous cations. All
of these results indicate that the selectivity of L1 for the Cu2+ ion
over other competitive cations in the water medium is remarkably
high.
Fig. 6 Confocal fluorescence images of Cu2+ in Hep G2 cells (Zeiss
LSM 510 META confocal microscope, 40¥ objective lens). (a) Brightfield
transmission image of Hep G2 cells. (b) Fluorescence image of Hep G2
cells incubated with L1 (5 mM). (c) Cells supplemented with 5 mM L1 in
the growth media for 0.5 h at 37 ◦C and then incubated with 10 mm CuCl2
for 2 h at 37 ◦C.
In conclusion, we have developed a new fluorescent sensor L1
based on the coumarin fluorophore. It shows high sensitivity and
selectivity toward Cu2+ in aqueous solution. The complexation
mode and corresponding quenching mechanism were elucidated
by Job plot and DFT calculations. Living cell image experiments
further demonstrated its value in the practical applications of
biological systems.
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Fig. 5 Fluorescence intensities of 5 mM L1 upon the addition of various
metal ions in H2O/DMSO (9 : 1, v/v) solution. Gray bars represent the
fluorescence response of L1 to the metal ion of interest (5 mM for all
other metal ions). Black bars represent the addition of Cu2+ (5 mM) to the
solution. (lex = 450 nm).
To test the sensitivity of the interaction between L1 and Cu2+,
we also investigated the time course of the response of L1 (5 mM)
in the presence of 1 equiv. of Cu2+ in H2O/DMSO (9 : 1, v/v). It
was found that the obvious spectral change was observed within
2 min upon addition of 1 equiv. Cu2+ (Fig. S14 ESI†). Therefore,
this system could be used for real-time tracking of Cu2+ in cells
and organisms.
Then, we studied bioimaging applications of L1 for monitoring
of Cu2+ ions in biological systems. We primarily carried out
experiments using L1 on liver cell lines in which Cu2+ is known
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