A highly selective and sensitive in vivo fluorosensor for zinc(ii) without cytotoxicity

Article abstract of DOI:10.1039/c2ob07084g

A highly selective and sensitive fluorescent Zn²⁺ sensor, 2,6-bis(2-hydroxy-benzoic acid hydrazide)-4-methylphenol (1), was designed and synthesized. In aqueous THF (4:6 v/v) ligand 1 induces a 2:1 complex formation with respect to Zn²⁺

Full text of DOI:10.1039/c2ob07084g

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COMMUNICATION
A highly selective and sensitive in vivo fluorosensor for zinc(II) without
cytotoxicity†
Tarun Mistri,a Malay Dolai,a Debrup Chakraborty,b Anisur Rahman Khuda-Bukhsh,b Kalyan Kumar Dasa
and Mahammad Ali*a
Received 13th December 2011, Accepted 16th January 2012
DOI: 10.1039/c2ob07084g
A highly selective and sensitive fluorescent Zn2+ sensor, 2,6-
must have the ability to be passively and irreversibly loaded into
cells. Though recently, there are significant advances in the
design of fluorescent probes for Zn2+,10 and many of them are
suitable for biological applications and have been used with
some success in Zn2+ neurobiology,11 however, arguably all
suffer from one or more limitations. For example, fluorescent
Zn2+ probe FluoZin-3, most frequently used to detect Zn2+, has
high sensitivity, quantum yield and high affinity for Zn2+ (Kd =
15 nM) at pH 7.412,13) but it has a drawback in its selectivity,
being fluorescent also towards Ca2+ particularly at higher
concentration.
The purpose of the present study is to design and synthesize a
simple and inexpensive zinc sensor with high selectivity and
sensitivity of nM range which could identify the in vivo and
in vitro level of [Zn2+] below 3 nM,14 and at the same time
would have no or negligible cytotoxicity.
Ligand 1 was prepared by simple Schiff base condensation of
4-methyl-2,6-diformylphenol(dfc) with salicylhydrazide in the
presence of a catalytic amount of AcOH. The Zn-complex was
prepared by the reaction of Zn-acetate and the ligand in MeCN.
Single crystals suitable for X-ray diffraction were obtained by
recrystallization from DMF. All these are presented in Scheme 1
and details of experimental procedures are presented in the ESI.†
Single crystal X-ray diffraction studies show that the complex
bis(2-hydroxy-benzoic acid hydrazide)-4-methylphenol (1),
was designed and synthesized. In aqueous THF (4 : 6 v/v)
ligand 1 induces a 2 : 1 complex formation with respect to
Zn2+ at physiological pH. This probe features visible light
excitation(390 nm) and emission (490 nm) profiles, excellent
selectivity responses for Zn2+over other competing biological
metal ions with Kd < 1 pM2, LOD < 1 ng L−1 and about 680
fold enhancement in fluorescent intensity upon Zn2+ binding.
It also exhibits cell permeability and intracellular Zn2+
sensing in A375 human melanoma cancer cell.
Zinc is the second most abundant transition metal in our body
after iron and it is essential for normal cellular functions leading
to normal human growth and development. It also serves an
additional signalling role in the Central Nervous System (CNS),1
which contains high levels of Zn2+,2 largely localized into the
synaptic vesicles of excitatory nerve terminals.3 The behavior of
several ion channels and receptors,4 like NMDA and potentiation
of AMPA receptors5 are affected on the release of Zn2+ from
nerve terminals after transient global ischemia6 sustained sei-
zures,7 and head trauma.8 However, little is known about the dis-
tribution, accumulation and mobility of intracellular zinc. The
major challenge is to apply a Zn-sensor to real-life situations. To
apply a luminescent probe to biomedical imaging, one needs to
assess them intracellularly and optimize the photophysical
properties.
ˉ
2 crystallizes in the space group P1(triclinic) as a dinuclear
species with a μ2-cresolato and two μ2-acetato bridge cores
(Fig.1). Both Zn atoms are five-coordinated with NO4 donor sets
Desirable intracellular fluorescent sensors should have: (i) a
signal with a high quantum yield (Φ) to permit the use of glass
microscope objectives, (ii) excitation wavelengths exceeding
340 nm to prevent UV-induced cell damage, (iii) emission wave-
lengths approaching 500 nm to avoid autofluorescence from
species native to the cell and to facilitate use with typical fluor-
escence microscopy optical filter sets9 and finally, (iv) sensors
aDepartment of Chemistry, Jadavpur University, Kolkata 700 032, India
bDepartment of Botany, Kalyani University, Kalyani, 700 032, India.
E-mail: m_ali2062@yahoo.com
†Electronic supplementary information (ESI) available: Details of exper-
imental procedures. CCDC reference numbers 834115. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/
c2ob07084g
Scheme 1
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made up of a bridging μ2-cresolato O atom, a salicylhydrazide N
atom, a salicylhydrazide O atom and the other two O atoms from
two different acetate ions. The O4 atom on the salicylhydrazide
part is enolic type and coordinated to Zn1, while the other sali-
cylhydrazide oxygen atom, O3 is ketonic type and coordinated
to Zn2. The τ values of 0.56 [Zn1] and 0.69 [Zn2] show that the
geometries at the metals are intermediate between trigonal-bipyr-
amid and square-pyramid (for a perfect square-pyramid, τ = 0
and for a perfect trigonal-bipyramid, τ = 1). The separation
between the two metal centers (Zn1⋯Zn2) is 3.182 Å. The other
bond distances are O4–Zn1 = 2.0499(15), O3–Zn2 = 2.1416
(17), N5–Zn1 = 2.0256(18) and N9–Zn2 = 2.0311(18) which are
the salicylhydrazide parts of the ligand. Two acetate bridges are
hanging from the two penta-coordinated Zn metals. These bond
distances are Zn1–O1 = 1.9751(17) Å, Zn1–O6 = 1.9802(18) Å
and Zn2–O2 = 1.9633(17) Å, Zn2–O5 = 1.9554(18) Å. The two
DMF (dimethyl formamide) molecules are also present in this
crystal lattice with one DMF molecule showing H-bonding inter-
action with free –OH group, situated at ortho-position of salicyl-
hydrazide with O9–H51⋯O11 H-bond distance = 1.83(3) Å.
The selected bond angles and bond distances are listed in the
Table S1.†
The spectrophotometric titration for the interaction of 1 with
Zn2+ at 25 °C in aqueous-THF buffer (HEPES buffer, pH 7.0)
solution (4 : 6 v/v) reveals that there are gradual increases in
absorption intensities of 1 at 434 and 350 nm and a decrease at
383 nm with isosbestic points at 390, 373 and 331 nm,
accompanied by the obvious hypsochromic shift of 476 nm
absorption peak of free ligand 1 to 434 nm, with the gradual
increase in the concentration of Zn2+ in the range 0–10.0 μM,
keeping the concentration of 1 fixed at 10.0 μM (Fig. 2(a)).
On further increase in concentration of Zn2+ the absorption
band at 434 nm gradually shifted towards 420 nm and 383 band
to 365 nm; and isosbestic points at 390 and 373 nm are shifted
towards 380 and 362 nm respectively (see Fig. S5a†).This may
arise due to the change in composition of 1 and Zn2+ from 1 : 1
to 1 : 2. While the isosbestic point at 331 nm for 1 : 1 compo-
sition vanishes and a new isosbestic point is generated at 450 nm
for [Zn2+] : [1] > 1. The blue shift in absorption bands of free
ligand is expected to correspond to the coordination of Zn2+
with 1 via nitrogen and oxygen atoms in the salicylhydrazide
moiety to generate a five-membered chelate ring; which conse-
quently extends the conjugation resulting in a shift of absorption
band at 460 nm to 426 nm. The appearance of three isosbestic
points also clearly indicates the transformation of 1 to the corre-
sponding Zn-coordinated species. The composition of the
complex was determined by JOB’s method (inset of Fig. 2(c))
and found to be 1 : 2 with respect to ligand.
DFT calculations on complex 2 were performed using Gaus-
sian 03 program15 starting from the X-ray coordinates. The
global minima of all these species were confirmed by the posi-
tive vibrational frequencies. The MO diagram of 2 is shown in
Fig. 3. Bond distances and bond angles around the Zn2+ centers
are in agreement with the crystallographic data (see Table S1†).
The UV spectrum computed from TDDFT calculations16,17 in
THF shows three important peaks in the range 270–450 nm (see
inset (d) in Fig. 2). The band around 450 nm is dominated by
Fig. 1 (a) ORTEP plot of compound 2 with atom numbering (30%
thermal ellipsoid probability); (b) DFT optimized structure of 2.
Fig. 2 (a) Change in absorption spectra of 1 (10 μM) upon addition of Zn2+ in HEPES buffer at pH 7.0 in H2O–THF = 4 : 6 v/v at 25 °C, [Zn2+] =
2–30 μM. Inset (b) plot of absorbance (at 426 nm) vs. [Zn2+]; (c) JOB’s plot for the same reaction at 426 nm and (d) TD-DFT spectrum of 2 in THF.
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Fig. 3 Frontier molecular orbitals of complex 2.
Fig. 5 Selectivity of 1 for Zn2+. Fluorescence response of 1 following
addition of 15 equivalents of the metal ion of interest. Inset is the fluor-
escence spectrum of 1 in the presence of 15 equivalents of different
metal ions.
enhancement is 150 times that observed in the case of ZQ1 and
ZQ210b In contrast, addition of other metal cations scarcely
shows any fluorescence enhancement (Fig. 5).The quantum
yield determination of the zinc complex gives Φ(2) = 0.15,
while the free ligand was found to be non- or very weakly fluor-
escent. The Limit of Detection (LOD) of Zn2+ was determined
by the 3σ method18 and found to be 2.88 ng L−1. All these
findings indicate that 1 behaves as a highly sensitive fluorescent
Zn2+ sensor. Addition of TPEN (N,N,N′,N′-tetra(2-picolyl)ethy-
lenediamine), an intracellular heavy metal ion chelator, to a
solution containing 1 and Zn2+ leads to the immediate disappear-
ance of the yellowish-green fluorescence. This indicates that 1
reversibly coordinates to Zn2+.
Fig. 4 Change in fluorescence intensity (λex 390 nm) of 1 (1.0 μM)
measured in H2O–THF = 4 : 6 v/v in HEPES buffer at pH 7.0, upon
addition of Zn2+ (0–2.20 μM); Inset: (a) plot of {(Fmax − Fo)/(F − Fo)}
vs. 1/[Zn2+]2: (b) plot of F (at 490 nm) vs. [Zn2+].
Fig. 4b shows the result of fluorescence titration of 1 with
Zn2+. There are two Zn2+ ions coordinated to one ligand frag-
ment and this is confirmed by the Benesi–Hildebrand method.
Assuming a 1 : 2 association between 1 and Zn2+, the Benesi–
Hildebrand equation is given as:
the HOMO(155) → LUMO(156) excitation, while the band
around 370 nm is mainly due to the HOMO(155) → LUMO + 1
(157) and HOMO − 1(154) → LUMO(156) transitions. The
details of the vertical excitation energies, oscillator strengths,
and nature of excitations are shown in Table S2.† The pattern of
the calculated UV-Vis spectrum is very close to the experimental
one.
Fig. 4 shows the change in fluorescence intensity at λem
490 nm (λex 390 nm) of 1 (1.0 μM) measured in THF–water
(4 : 6 v/v) with successive addition of Zn2+ (0.05–2.2 μM) at
physiological pH (7.0, HEPES buffer). The addition of Zn2+
leads to an increase in fluorescence intensity at around 490 nm
and it is saturated upon addition of 2.20 equivalent of Zn2+. The
fluorescence increase occurs immediately upon addition of Zn2+.
There is a gradual shift in λem from 512 nm to 486 nm that may
be attributed to the fact that on coordination to Zn2+ through
deprotonated cresolato and enolato oxygen atoms of the ligand
leads to a decrease in the polar environment of the ligand.
As shown in Fig. 4, the fluorescence enhancement factor
measured at 490 nm is determined to be ∼680 at [1] = 1.0 μM
and [Zn2+] ≥ 2.0 μM and to the best of our knowledge this
seems to be the highest value among the previously reported
related Zn-sensors.10 So far the highest known fluorescence
ðFmax ꢀ F0Þ
ðF ꢀ F0Þ
1
¼ 1 þ
2
K½Zn2þꢁ
where, F0 is the fluorescence of 1 in the absence of externally
added Zn2+, F is the fluorescence obtained at different [Zn2+]
and Fmax is the fluorescence of 1 at [Zn2+] in large excess. K
(M−2) is the association constant. As shown in Fig. 4(b), the plot
of {(Fmax − F0)/(F − F0)} vs. 1/[Zn2+]2 yields a straight line
with slope = (8.51 0.06) × 10−13, indicating that 1 indeed
associates with Zn2+ in a 1 : 2 stoichiometry. The intercept value
1.07 0.48, close to 1.0, also manifests the self-consistency of
the experimental data. Therefore, the ligand dissociation constant
Kd is ∼0.85 pM2. The 1 : 2 complex formation in solution is
further confirmed by ESI-MS+-(m/z) analysis with a peak at
887.65 that corresponds to the composition {[Zn2(1)
(CH3COO)2]·(NMe2CHO)2 CH3CN + Na+}.
Zinc detection was not perturbed by biologically abundant
Li+, Na+, K+, Mg2+, Ca2+ etc. metal ions. Several transition
metals, namely Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cd2+,
Pb2+, and Hg2+, also caused no interference (Fig. 5).
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the ability of the probe to sense Zn2+ inside the cell of intracellu-
lar concentration (∼3 ng L−1). The cells also exhibited more
intense fluorescence when Zn2+ was introduced onto the cells
externally, and fluorescence responses increase with the increase
in Zn2+ concentration, which could be evident from the cellular
imaging (Fig. 6c–e). However, the fluorescence of the cells was
deeply suppressed when the inhibitor, TPEN, of the Zn2+ sensor
was added onto the cells (Fig. 6f–h). These observations clearly
indicate that the present zinc sensor is an efficient candidate for
monitoring changes in the intracellular Zn2+ concentration under
biological conditions. Fluorescence intensities of cells of differ-
ent experimental groups were analyzed and have been addition-
ally incorporated as Fig. S10.†
In order to test the cytotoxicity of 1 on the cell, we also per-
formed MTT assay19 in A375 human melanoma cancer cells
treated with various concentrations of zinc sensor for up to 2 h
(Fig. 7). But the result showed no significant cell death in 2 h
incubation of 1 up to 100 μM. This would therefore suggest that
this zinc sensor can readily be used for cellular application at the
indicated dose and time of incubation without worry about its
cytotoxicity.
To confirm our findings, we also subjected HeLa cells (a
human cervical carcinoma cell line) to the same type of treat-
ments and the results we obtained were essentially similar in
nature. Therefore, its use in biological materials as an effective
zinc monitoring candidate was further confirmed.
In summary, we have developed a simple and inexpensive
zinc sensor that features visible excitation and emission profiles,
excellent selectivity responses for Zn2+ over other competing
physiological metal ions at intracellular concentrations, a dis-
sociation constant Kd < 1 pM2, and a ∼680-fold increase in flu-
orescence emission intensity upon zinc binding leading to LOD
∼1 nM without requiring microinjection for intracellular use.
Fig. 6 Fluorescence microscopic observations: (a) Phase contrast and
(b) fluorescence images of A375 cells induced by intracellular Zn2+
when incubated with 10 μM zinc sensor for 30 min at 37 °C and washed
with PBS. Cells were exposed to sequentially increasing concentrations
of added extracellular Zn2+ as (c) 10 μM, (d) 20 μM and (e) 30 μM. The
return of intracellular Zn2+ to the resting level (f–h) was achieved by the
addition of zinc sensor inhibitor (TEPN, 100 μM), at 5 min interval. For
all imaging, the samples were excited at ∼360 nm.
Acknowledgements
Financial support from CSIR (Ref No. 01(2490)/11/EMR-II) and
DST (Ref. SR/S1/IC-35/2006), New Delhi, India are gratefully
acknowledged. T.M. acknowledges the fellowship from UGC
(UGC-NET).
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