An NBD fluorophore-based sensitive and selective fluorescent probe for zinc ion

Article abstract of DOI:10.1039/b712377a

A novel NBD-derived fluorescent probe for Zn²⁺ is described; the probe features ready availability, good water solubility, high sensitivity and selectivity, and ability to quantify the concentration of Zn²⁺. The Royal Society of Chemistry.

Full text of DOI:10.1039/b712377a

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COMMUNICATION
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An NBD fluorophore-based sensitive and selective fluorescent probe for
zinc ion{
Wei Jiang,a Qingquan Fu,a Hongyou Fanb and Wei Wang*a
Received (in Austin, TX, USA) 13th August 2007, Accepted 24th October 2007
First published as an Advance Article on the web 9th November 2007
DOI: 10.1039/b712377a
A novel NBD-derived fluorescent probe for Zn2+ is described;
problems such as poor water solubility, inadequate selectivity,
insufficient sensitivity and the lack of ability to quantitatively
measure Zn2+. In this communication, we wish to report a newly
designed NBD based fluorescent probe for Zn2+ based on the
photoinduced electron transfer (PET) mechanism. The probe
displays high selectivity and sensitivity toward Zn2+ ion and good
water solubility, demonstrating its potential for biological imaging.
More importantly, the chemosensor allows easy quantification of
Zn2+ concentration.
the probe features ready availability, good water solubility, high
sensitivity and selectivity, and ability to quantify the concentra-
tion of Zn2+.
Zinc ion (Zn2+), the second most abundant transition metal in the
human body, plays myriad roles in numerous cellular functions
such as the regulation of gene expression, apoptosis, co-factors in
metalloenzyme catalysis, and neurotransmission in biological
systems.1 Deregulation of Zn2+ is implicated in several diseases
including Alzheimer’s disease,2 prostate cancer,3 and diabetes.4
Accordingly, the development of Zn2+-specific molecular probes
has been of considerable interest in the areas of chemical and
biological sciences.
The PET mechanism has been widely exploited for molecular
sensing.15 In the design of PET fluorescent sensors for Zn2+, the
critical issue is that Zn2+ binding to the sensor should generate a
detectable signal so that the binding event can be monitored
conveniently. We envisioned that chelation of Zn2+ with
compounds 1 or 2 would cause the fluorescence intensity to
increase as a result of blocking PET of the nitrogen atoms.
Accordingly, the newly designed NBD fluorescent probes for Zn2+
are composed of two essential components: an NBD moiety as a
reporter and N,N,N9,N9-tetrakis(2-pyridylmethyl)ethylenediamine
(TPEN) or N,N-bis(pyridin-2-ylmethyl)amine (BPA) as chelators
for Zn2+ (Fig. 1). TPEN and BPA are the chelators of choice since
they display high specificity for binding to Zn2+ over other metal
cations, and favorable kinetic and thermodynamic properties
which result in quick formation of a stable Zn2+ complex.9c–f,13
Furthermore, they are readily incorporated into a fluorophore.
The two moieties are directly tethered together through a robust
C–N bond without a linker (Fig. 1).
An ideal Zn2+ chemical probe with potential for biological
applications should possess: 1) good water solubility, 2) the
capability to quantitatively determine Zn2+ concentration, 3) long
excitation wavelength to avoid cell damage, 4) high stability, 5)
high selectivity and sensitivity toward Zn2+
, and 6) easy
preparation. The development of fluorescent chemosensors for
probing Zn2+ has been an active topic as a result of operational
simplicity and high sensitivity. However, the search for readily
accessible fluorescent Zn2+ probes with good water solubility and
high specificity is still a challenging task. It is a particular challenge
to develop a chemosensor which makes it possible to determine the
concentration of Zn2+ and to discriminate Zn2+ from Cd2+ owing
to their closely related properties. Fluorescent probes for sensing
Zn2+, based on various fluorophores such as quinoline,5 bipyridyl,6
dansyl,7 ferrocene,8 fluorescein,9 anthracene,10 benzofuran and
benzoxazole,11 naphthalimide12 and cyanine13 have been reported.
The bipyridyl,6 dansyl,7 ferrocene,8 anthracene10 and naphthali-
mide12 based probes have poor water solubility. Generally a
mixture of organic solvent and water is used, thus limiting their
biological applications. The widely used quinoline5 and fluor-
escein9 derived chemosensors provide good solubility; however,
their selectivity for Zn2+ and Cd2+ is not clear. Moreover, the
syntheses are not trivial. However, to the best of our knowledge, a
7-nitrobenz-2-oxa-1,3-diazole (NBD) derived fluorescent Zn2+
chemical probe has not been described, despite the fact that it
has been widely used in molecular imaging in biological systems.14
Moreover, some of these reported fluorescent probes suffer from
Sensors 1 and 2 were synthesized in a straightforward manner
(see ESI{). The investigation of the fluorescence properties of
probes 1 and 2 reveals that free 1 exhibits very weak fluorescence
(Fig. 2), while 2 displays a strong signal (Fig. S1 in ESI{). Upon
addition of Zn2+, the fluorescence intensity of probe 1 is enhanced
significantly in a concentration dependent manner (Fig. 2).
Moreover, when ¢1.0 equiv. of Zn2+ is added, the maximum
aDepartment of Chemistry & Chemical Biology, University of New
Mexico, Albuquerque, NM 87131, USA. E-mail: wwang@unm.edu;
Fax: (+1) 505 277 2609; Tel: (+1) 505 277 0756
bCeramic Processing and Inorganic Materials Department, 01815,
Sandia National Laboratories, Albuquerque, NM 87185, USA
{ Electronic supplementary information (ESI) available: Experimental and
NMR data. See DOI: 10.1039/b712377a
Fig. 1 Design of NBD-derived fluorescent probes for Zn2+
.
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Fig. 2 Emission spectra (lex = 470 nm) of probe 1 (1025 M) after
addition of a range of amounts of Zn2+ (0–1.4 6 1025 M) at room
temperature in PBS buffer (pH 7.3).
Fig. 4 The selectivity of probe 1 toward Zn2+ and other metal ions. In
these experiments, the fluorescence measurement was taken at lex
=
470 nm from 1025 M of probe 1 in a PBS buffer (pH 7.3) at room
temperature and in the absence and presence of 1.0 equiv. of a metal ion.
The fluorescence intensity at lem = 550 nm is used for plotting versus an
analyte.
fluorescence intensity is reached. The intensity is increased by more
than 25-fold. This indicates the probe is highly sensitive to Zn2+
with a Kd of 4.6 mM (see ESI{). Furthermore, as expected, the
sensor 1 forms a 1 : 1 complex with Zn2+ (see ESI{).
More significantly, a linear relationship between the fluorescence
intensity of probe 1 and the concentration of Zn2+ is observed
(Fig. 3). Therefore the sensor could be used for the quantitative
determination of the concentration of Zn2+. In contrast, no
fluorescence alternation for sensor 2 is observed (Fig. S1 in ESI{).
This indicates that the ‘‘N1’’ in probe 1 is critical in the PET
(Fig. 1).
enhancement is achieved. Since the concentration of Zn2+ in a
biological system, for example, in synaptic vesicles, is reported to
be in the micro- to millimolar range,16 the probe 1, which displays
a sensitivity in the micro-range, can be used for the imaging of
Zn2+. Moreover, the magnitude of the fluorescence intensity
increase corresponds nearly linearly to the concentration of Zn2+
,
indicating that the sensor could be used for the quantitative
measurement of Zn2+ concentrations.
The above studies prompt us to select chemical probe 1 for
further evaluation aimed at determining its selectivity. The
fluorescence titration of 1 with various metal ions exhibits high
selectivity to Zn2+ (Fig. 4). Metal ions which possess a broad
spectrum of biological activities and functions in living cells, such
as Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, and Fe3+, do not give rise to
any responses under the same conditions. Most heavy transition
metal ions, including Cd2+, Ni2+, and Co2+, also show no
interference. Hg2+ induces very limited fluorescence enhancement,
while Cu2+ quenches fluorescence.
Financial support for this work provided by the Department of
Chemistry & Chemical Biology and the Research Allocation
Committee, the University of New Mexico, NIH-INBRE (P20
RR016480), and the Sandia University Research Program (SURP)
is gratefully acknowledged.
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In conclusion, a novel NBD-derived water-soluble fluorescent
chemical probe has been designed and synthesized, and it displays
high selectivity and sensitivity for Zn2+ in a neutral buffer aqueous
solution. In the presence of Zn2+
, significant fluorescence
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Fig. 3 Plot of the concentration of Zn2+ vs. DI/I0, where DI = I 2 I0, I:
the fluorescence intensity of probe 1 (1025 M) with addition of Zn2+ and
I0: the fluorescence intensity of probe 1 without Zn2+ at lem: 550 nm.
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