Structurally Characterized Zn2+ Selective Ratiometric Fluorescence Probe in 100 % Water for HeLa Cell Imaging: Experimental and Computational Studies

Article abstract of DOI:10.1007/s10895-015-1688-9

Fluorescence recognition of Zn²⁺ in 100 % aqueous medium using 2-((1, 3 dihydroxy-2-(hydroxymethyl)propan-2 ylimino) methyl) phenol (SALTM) as ratiometric probe is reported. Moreover, SALTM can discriminate Zn²⁺ from Cd²⁺

Full text of DOI:10.1007/s10895-015-1688-9

J Fluoresc
DOI 10.1007/s10895-015-1688-9
ORIGINAL ARTICLE
2+
Structurally Characterized Zn Selective Ratiometric
Fluorescence Probe in 100 % Water for HeLa Cell Imaging:
Experimental and Computational Studies
1
1
1
1
2
Babli Kumari & Sisir Lohar & Milan Ghosh & Sabyasachi Ta & Archya Sengupta
&
2
2
1
Prajna Paramita Banerjee & Ansuman Chattopadhyay & Debasis Das
Received: 13 June 2015 /Accepted: 7 October 2015
#
Springer Science+Business Media New York 2015
2
+
Abstract Fluorescence recognition of Zn in 100 % aqueous
and immune function [1, 2]. In addition, disruption of
2
+
medium using 2-((1, 3 dihydroxy-2-(hydroxymethyl)propan-
ylimino) methyl) phenol (SALTM) as ratiometric probe is
reported. Moreover, SALTM can discriminate Zn from Cd
very effectively. The binding constant and detection limit of
the probe for Zn is 2.2×10 M and 2.79×10 M respec-
tively. Interestingly, corresponding naphthalene derivative
Zn homeostasis may be implicated in a number of
severe neurological diseases like Alzheimer’s &
Parkinson’s diseases, amyotrophic lateral sclerosis, hyp-
oxia ischemia, and epilepsy [2–13]. Therefore, recogni-
2
2
+
2+
2
+
4
-1/2
−8
2+
tion and determination of Zn in living tissues is a
subject of great importance [1, 2, 14] and attracted im-
mense interest in chemical and biological sciences.
(
HNTM) having less water solubility fails to be a ratiometric
2
+
2+
sensor. SALTM can detect intracellular Zn in HeLa cervical
cancer cells under fluorescence microscope. Moreover, DFT
and TD-DFT studies support experimental findings.
Fluorescence recognition of Zn has paramount impor-
tance as it does not provide any spectroscopic or mag-
netic signals [2, 15–18]. According to the World Health
Organization (WHO), the maximum acceptable level of
2
+
.
.
.
Zn in drinking water is 76 μM. Hence, selective rec-
Keywords Zn2+ Ratiometric Fluorescence HeLa cell
2
+
.
ognition of Zn is very important in supramolecular
chemistry [19]. Recently, considerable efforts have been
imaging Computational studies
2
+
observed for in vitro and in vivo detection of Zn
20–24]. Most of them are based on quinoline, anthra-
cene, coumarin, BODIPY, and fluorescein fluorophores
25–32]. However, most of them involve complicated
[
Introduction
[
As second most abundant metal ion in human body
after iron, zinc plays vital role in numerous biological
processes, including brain activity, gene transcription,
syntheses/hazardous reaction conditions and expensive
chemicals/reagents. Some have poor water solubility/ in-
terference from same group cations [33–36], limiting
2
+
their real applications. Thus, interference free Zn sen-
sor that functionsin fully aqueous medium [37] for real
applicationsis highly demanding. Schiff bases [31, 32,
CCDC numbers of SALTM and HNTM are 992217 and 993440
respectively.
3
8–40] incorporating a fluorescent unit are appealing
*
Debasis Das
ddas100in@yahoo.com
reagents for optical sensing of metal ions [38–42]. Fur-
thermore, most of the sensors exhibit only changes of
emission intensity at a single wavelength which may be
misleading due to several environmental factors. This
may be eliminated through ratiometric fluorescence
sensing and hence, more desirable [43, 44]. All these
facts have inspired us to designa very simple, highly
1
2
Department of Chemistry, The University of Burdwan,
Burdwan, West Bengal, India
Department of Zoology, Visva Bharati University, Santiniketan, West
Bengal, India

J Fluoresc
Scheme 1 Synthesis of SALTM
and HNTM
2
+
sensitive and Zn selective ratiometric fluorescence sen-
sor, SALTM that operates in 100 % aqueous solution.
SALTM is prepared from salicyldehyde and
tris(hyrdoxymethyl)amino methane as shown in
Scheme 1. Replacing salicyldehyde by 2-hydroxy
naphthaldehyde generates a model compound, HNTM
that does not behave ratiometrically, however normal
2
+
turn on Zn sensing is observed. A similar probe to
that of HNTM where amino methanol units are replaced
by aminoethanol units, also functions as a normal turn
2
+
Fig. 1 Single crystal X-ray structure of SALTM
on Zn sensor [45]. To the best of our knowledge,
1
Fig. 2 H NMR spectrum of
SALTM in DMSO-d
6

J Fluoresc
Fig. 3 QTOF mass spectrum of SALTM
2
+
SALTM is the first ratiometric Zn sensor in 100 %
water. HeLa cell comprises an immortalized, continuous-
ly cultured cell line of human cancer cells. Unlike nor-
mal body cells, HeLa cells thrive indefinitely in labora-
tory tissue culture, a trait that has made them important
in research work. Fluorescence cell imaging of intracel-
Solvents used are of spectroscopic grade. All other
chemicals are of analytical grade and used without
further purification. Mili-Q Milipore® 18.2 MΩ cm
−
1
water is used in experiments. Shimadzu UV–Vis-2450
spectrophotometer is used for recording absorption
spectra. FTIR spectra are recorded on a Shimadzu
FTIR spectrometer. Mass spectra are performed on a
QTOF Micro YA 263 mass spectrometer in ES posi-
tive mode [1].H NMR spectra are recorded with
Bruker Avance 600 (600 MHz) spectrometer. Solution
pH is measured with Systronics digital pH meter
(model 335). HCl or NaOH (50 mM) is used for pH
adjustment. The steady-state fluorescence spectra are
recorded with a Hitachi F-4500 spectrofluorimeter.
The fluorescence microscope imaging system is com-
posed of an inverted fluorescence microscope
(Dewinter, Italy). Images are captured with an attached
2
+
lular Zn is performed in HeLa cells [46, 47].
Experimental
General Procedure
High-purity HEPES, tris(hydroxylmethyl)amino meth-
ane, salicyldehyde and 2-hydroxy napthaldehyde have
been purchased from Sigma Aldrich (India).
Zn(OAc) .2H O is purchased from Merck (India).
2
2

J Fluoresc
90
%T
75
6
4
3
1
0
5
0
5
4
000
3500
3000
2500
2000
1750
1500
1250
1000
750
500
1/cm
Fig. 4 FTIR spectrum of SALTM
CCD camera and processed with BioWizard 4.2 soft-
ware. The microscope is equipped with a 50 W mer-
cury arc lamp.
tris(hydroxymethyl)amino methane (248 mg, 2.05 mmol,
25 mL) in methanol. The mixture is refluxed for 5 h. Slow
evaporation of the solvent produces yellow crystals of the
receptor in 91 % yield (420 mg). The structure of the
receptoris established by single crystal X-ray structure
Synthesis of 2-((1, 3-dihydroxy-2-(hydroxymethyl)propan-
1
(
Fig. 1). H NMR (Fig. 2,) (600 MHz, DMSO-d ), δ (ppm):
6
2
ylimino)methyl)phenol(SALTM) SALTM is synthesized as
shown in Scheme 1. Methanol solution of salicylcadehyde
2 5 0 m g , 2 . 0 5 m m o l , 1 0 m L ) i s a d d e d t o
8
7
.52 (1H, S), 7.37 (1H, d, J=2.4Hz),7.35 (1H, t, J=1.2Hz),
.23 (2H, m, J=10.8Hz), 3.57 (6H, d, J=8.4); ESI MS
(
+
−1
(Fig. 3): [SALTM+H] =226.2; FTIR (cm ) (Fig. 4): υ(O-
H phenol) 3313, υ(C=O ketone) 1631, υ(CH=N) 1595.
Synthesis of 1-((1,3dihydroxy-2-(hydroxymethyl)propan-
2
-ylimino)methyl)naphthalene-2-ol(HNTM) Scheme 1
shows the synthetic pathway for HNTM. Methanol so-
lution of 2-hydroxy napthaldehyde (250 mg, 1.45 mmol,
1
0 mL) is added to dry methanol solution of
tris(hydroxymethyl)amino methane (176 mg, 1.45 mmol,
5 mL). The mixture is refluxed for 5 h. Slow evapo-
2
ration of the solvent generates yellow crystals of
HNTM in 87 % yield (350 mg). The structure of
HNTM has been established by single crystal X-ray
strcuture (Fig. 5).
Fig. 5 Single crystal X-ray structure of HNTM

J Fluoresc
1
05
%
T
0
9
7
6
4
3
5
0
5
0
4
000
3500
3000
2500
2000
1750
1500
1250
1000
750
500
/cm
1
2+
Fig. 6 FTIR spectrum of SALTM-Zn complex
2
+
Synthesis of [Zn -SALTM-H]adduct
2013) program. The structure is solved by standard di-
rect methods [48] and refined by full matrix least
squares on F [49].
2
1
0
0 mL methanol solution of Zn(OAc) .2H O (87.6 mg,
.4 mmol) is added slowly to the magnetically stirred
2
2
solution of SALTM (90 mg, 0.4 mmol, 10 mL) in dry
methanol. The mixture is stirred for 30 min and kept at
room temperature for few days to get white crystalline
Cytotoxicity Assay
In vitro cytotoxicity is measured using the colorimetric
methyl thiazolyltetrazolium (MTT) assay against Hella
cells. Cells are seeded into 24-well tissue culture plate
in presence of 500 mL Dulbecco’s modified eagle me-
dium (DMEM) supplemented with 10 % fetal bovine
serum (FBS) and 1 % penicillin/streptomycin at 37 °C
2
+
−1
[
SALTM-Zn ] adduct. FTIR (cm ), (Fig. 6): υ(O-H,
phenol), 3340; υ(C=O, ketone), 1625, υ(C=N), 1595.
2
+
The ESI-MS spectrum (Fig. 7) of [SALTM-Zn ] adduct
shows peak at m/z, 513 which clearly confirms the ad-
duct formation.
and 5 % CO atmosphere for overnight and then incu-
2
Single Crystal X-ray Structure Analysis
bated for 48 h in presence of SALTM at different con-
centrations (10–100 mM). Then cells are washed with
PBS buffer and 500 mL supplemented DMEM medium
is added. Subsequently, 50 mL MTT (5 mg mL ) is
added to each well and incubated for 4 h. Next, violet
formazan is dissolved in 500 mL sodium dodecyl sul-
fate solution in water–DMF mixture. The absorbance
of solution is measured at 558 nm using microplate
reader. The cell viability is determined assuming
100 % cell viability for cells without SALTM.
Diffraction data for SALTM and HNTM are collected at
−
1
2
96[2] K on a Bruker SMART 1000 CCD diffractome-
ter using graphite-monochromated Mo-Kα radiation (λ=
.71073 Å). Some significant crystal parameters and re-
0
finement data are summarized in Tables 1, 2, 3, 4, 5
and 6. Data are processed and corrected for Lorentz
polarization effects. Multi-scan absorption corrections
have been performed using SHELXL-2013(Sheldrick,

J Fluoresc
2+
Fig. 7 QTOF mass spectrum of [SALTM-Zn ] complex
Results and Discussion
Table 1 Crystal parameters for SALTM
Identification code
Chemical formula
Formula weight
Temperature
SALTM
SALTM and HNTM have been synthesized following
Schemes 1 and 2. Single crystals of SALTM and
HNTM are grown from methanol solution and
11 4
C H15NO
225.24
296(2) K
0.71073 Å
Wavelength
Crystal system
Space group
Monoclinic
P 1 21/c 1
Table 2 Bond lengths (Å) of SALTM
O(001)-C(007)
O(003)-C(00D)
N(004)-C(009)
C(006)-C(00A)
C(006)-C(007)
C(009)-C(00C)
C(009)-C(00B)
C(00E)-C(00G)
1.2970(18)
1.417(2)
O(002)-C(00B)
N(004)-C(008)
O(1)-C(00C)
1.4141(18)
1.2978(19)
1.419(2)
1.423(2)
1.418(2)
1.538(2)
1.365(2)
1.372(3)
Unit cell dimensions
a=10.5261(4) Å, b=8.7390(3)
Å, c=12.6573(4) Å
α=90°, β=101.6120(15)°, γ=90°
1.4790(17)
1.415(2)
3
Volume
1140.48(7) Å
C(006)-C(008)
C(007)-C(00 F)
C(009)-C(00D)
C(00A)-C(00E)
C(00 F)-C(00G)
Z
4
1.4329(19)
1.5285(19)
1.539(2)
3
Density (calculated)
Absorption coefficient
F(000)
1.312 g/cm
0.100 mm−
1
480
1.402(3)

J Fluoresc
Table 3 Bond angles (°) for
SALTM
C(008)-N(004)-C(009)
C(00A)-C(006)-C(007)
O(001)-C(007)-C(00 F)
C(00 F)-C(007)-C(006)
N(004)-C(009)-C(00C)
C(00C)-C(009)-C(00D)
C(00C)-C(009)-C(00B)
C(00E)-C(00A)-C(006)
O(1)-C(00C)-C(009)
127.70(12)
119.99(13)
122.19(13)
116.64(13)
111.95(12)
108.06(13)
113.14(12)
121.54(14)
114.08(13)
118.73(15)
121.53(15)
C(00A)-C(006)-C(008)
C(008)-C(006)-C(007)
O(001)-C(007)-C(0060
N(004)-C(008)-C(006)
N(004)-C(009)-C(00D)
N(004)-C(009)-C(00B)
C(00D)-C(009)-C(00B)
O(002)-C(00B)-C(009)
O(003)-C(00D)-C(009)
C(00G)-C(00 F)-C(007)
118.93(13)
121.03(13)
121.17(13)
123.38(13)
106.08(11)
106.58(11)
110.82(12)
111.50(12)
112.29(13)
121.51(15)
C(00A)-C(00E)-C(00G)
C(00 F)-C(00G)-C(00E)
characterized by X-ray crystallography (Figs. 1 and
). The most significant geometric data are shown in
Tables 1, 2, 3, 4, 5 and 6.
2
Table 4 Crystal parameters of HNTM
Identification code
HNTM
As pH of the medium may affect the charge distri-
bution of the receptor, specially having acidic donor
site, and consequently influence its indigenous emission
properties, optimization of pH have been performed to
monitor the emission characteristics of SALTM and its
Chemical formula
Formula weight
Temperature
15 4
C H17NO
275.29
296(2) K
0.71073 Å
Wavelength
2+
Zn complex.
Crystal system
Space group
Monoclinic
P 1 21/c 1
Figure 8 shows poor sensing abilities of SALTM
2+
for Zn at pH below 6, probably due to protonation
Unit cell dimensions a=21.131(5) Å, b=5.9906(12) Å, c=10.662(2)
Å
of the donor centers. However, SALTM recognizes
2+
Zn
over a wide range of pH from 6.0 to 11.0.
α=90°, β=92.077(15)°, γ=90°
Two pKa [50] values for SALTM, viz. 5.06 (for
3
Volume
Z
1348.8(5) Å
4
3
Density (calculated) 1.356 g/cm
1
Table 6 Bond angles (°) of HNTM
Absorption
coefficient
F(000)
0.099 mm−
C(007)-N(005)-C(006) 126.9(4) N(005)-C(006)-C(00A) 113.9(4)
N(005)-C(006)-C(008) 105.7(4) C(00A)-C(006)-C(008) 111.4(4)
N(005)-C(006)-C(00B) 106.6(4) C(00A)-C(006)-C(00B) 107.5(4)
C(008)-C(006)-C(00B) 111.7(4) N(005)-C(007)-C(009) 123.3(5)
O(002)-C(008)-C(006) 109.8(4) C(007)-C(009)-C(00E) 119.2(4)
584
Table 5 Bond lengths (Å) of HNTM
O(001)-C(00B)
O(003)-C(00E)
N(005)-C(007)
C(006)-C(00A)
C(006)-C(00B)
C(009)-C(00E)
C(00C)-C(00D)
C(00C)-C(00H)
C(00E)-C(00I)
C(00G)-C(00 K)
C00J-C00K
1.413(7)
1.413(7)
1.304(6)
1.523(7)
1.540(7)
1.429(7)
1.412(7)
1.438(8)
1.445(7)
1.363(9)
1.375(8)
O(002)-C(008)
O(004)-C(00A)
N(005)-C(006)
C(006)-C(008)
C(007)-C(009)
C(009)-C(00D)
C(00C)-C(00G)
C(00D)-C(00 F)
C(00 F)-C(00 J)
C(00H)-C(00I)
1.427(6)
1.423(6)
1.486(6)
1.532(7)
1.410(7)
1.454(7)
1.418(7)
1.367(7)
1.367(7)
1.348(8)
2
+
Scheme 2 Proposed binding mechanism of SALTM to Zn

J Fluoresc
Fig. 8 Effect of pH on the emission intensities of SALTM (20 μM) and
2+
SALTM-Zn adduct
Fig. 10 Determination of pKa2 of SLTM using fluorescence method
(λEm, 450 nm)
pKa1 at acidic pH, Fig. 9) and 7.59 (for pKa2 at
basic pH, Fig. 10) indicate that its sensing ability is
higher around neutral pH. However, difference in the
emission intensities between SALTM and its Zn ad-
duct is highest at pH 7.4. SALTM shows weak emis-
2
+
change in emission properties is observed in presence of
2
+
2+
Zn (Fig. 11). Addition of Zn to the aqueous solution
of SALTM reduces the emission intensity at 370 nm
while a sharp fluorescence enhancement is observed at
450 nm. Thus a ratiometric fluorescence change is ob-
served with a sharp iso-emissive point at 381 nm, dem-
onstrating the existence of equilibrium in solution
sion centered at 370 nm (λ =300 nm) with a low
ex
quantum yield, 0.012. In presence of different com-
+
+
2+
2+
2+
2+
mon cations, viz. Na , K , Ca , Mn , Mg , Hg ,
2
+
2+
2+
2+
2+
2+
2+
Co
Ni
Cu , Pb , Fe , Zn and Cd , significant
,
,
(
Fig. 12). The intensity ratio, F450 nm/ F
exhibits
370 nm
2
+
a sigmoid curve against the added Zn (Fig. 13a). The
fluorescence quantum yield of the adduct increases more
than 6-fold compared to that of free probe having value,
0
.073.
SALTM has another excitation at 370 nm. Interest-
ingly, excitation at this wavelength in presence of
2
+
Zn
50 nm. As Zn selective ratiometric fluorescence
probe functioning in fully aqueous medium is very
results normal fluorescence enhancement at
2
+
4
2
+
rare, we have opted the ratiometric protocol for Zn
sensing.
Calculation of Quantum Yield
Fluorescence quantum yields (Ф) are estimated by inte-
grating the area under the fluorescence curves using the
equation,
ODstandard  Asample
Fig. 9 Determination of pKa1 of SALTM using fluorescence method
Em, 450 nm)
ϕsample
¼
 ϕstandard
ODsample  Astandard
(λ

J Fluoresc
2
+
2+
2+
2+
2+
Fig. 11 Relative emission intensity of SALTM (20 μM) in presence of
presence of different cations; Mn (1), Cd (2), Ni (3), Co (4), Mg
2+
2+
2+
2+
2+
3+
2+
different metal ions (100 μM) in aqueous solution(0.1 M, pH 7.4), λex
=
(5) Zn (6), Hg (7), Cu (8), Ca (9), Pb (10), Fe (11), Fe (12),
2+
3
00 nm, λem=450 nm (bottom). UV light exposed color of SALTM in
Pb (13) (top)
where A is the area under the fluorescence spectral
curve and OD is optical density of the probe at the
excitation wavelength [51]. Anthracene is used as
quantum yield standard (quantum yield 0.27 ethanol)
[52] for measuring the quantum yields of the probe
and its Zn complex.
2
+
Plot of the emission intensity vs. externally added
2
+
Zn generates a sigmoid graph (Fig. 13b) having line-
2
+
arity up to 100 μM Zn , useful for determination of
2
+
unknown Zn concentration. The fluorescence enhance-
ment is attributed to the formation of a rigid system
2
+
upon binding of the probe to Zn , restricting CH=N
isomerization leading to chelation-enhanced fluorescence
(
CHEF).
The detection limit [53] is calculated from the fluo-
rescence titration (Fig. 14). To determine the S/N ratio,
2
+
the emission intensity of SALTM without Zn is re-
peated 10 times from which the standard deviation of
blank is determined. The detection limit is then calcu-
lated using the equation: DL=3×SD/S, where SD is the
standard deviation of the blank and S is the slope of the
calibration curve. From the graph, the values for Sand
SD are 2.7484is 0.0256. Thus, the detection limit (DL)
−
8
is 2.79×10 M.
Corresponding naphthalene derivative, the model com-
pound, HNTM has low water solubility and recognize
Zn via normal turn on mechanism. A similar Zn sen-
sor to that of HNTM have reported the same observa-
tions [45].
Fig. 12 Changes in the emission spectra of SALTM (20 μM) in 100 %
water at pH 7.4 upon gradual addition of Zn (0.1, 0.2, 0.5, 1.0, 2.0, 3.0,
2+
2
+
2+
4
.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 25.0, 50.0, 100.0, 200.0, 400.0, 600.0 and
8
00.0 μM), λex=300 nm. (Inset: UV light exposed color change of
2+
SALTM upon addition of Zn

J Fluoresc
2
+
Fig. 14 Emission intensity vs. externally added Zn of SALTM (λEx
=
3
00 nm) for calculation of detection limit
2
+
constant, C is the concentration of Zn and n is the
2
+
number of Zn ions bound per SALTM molecule (here,
n=1/2) (Fig. 17).
On the other hand, fluorescence titration of HNTM
2
+
with Zn (Fig. 18) in HEPES buffer (pH 7.4, λ =
ex
3
80 nm, λem=450 nm) indicates normal turn on fluores-
cence behaviour.
The absorbance of aqueous solution of SALTM
(
pH 7.4) at 400 nm (Fig. 19) gradually decreases upon
Fig. 13 a Ratiometric calibration curve, I450 nm /I370 nm as a function of
2+
Zn concentration (λEx=300 nm). b Emission intensity vs. externally
2+
added Zn (0.1–800.0 μM) of SALTM (λEx=300 nm)
Job’s plot (Fig. 15) shows the stoichiometry of
2
+
SALTM –Zn complex as 2: 1 (mole ratio). A compet-
itive experiment with different metal ions indicates that
most of the 3d and bio-relevant cations do not interfere
significantly (Fig. 16). The binding constant of SALTM
2
+
4
−1/2
for Zn
is 2.2×10
M
, obtained using Benesi-
n
Hildebrand equation [54], Flim-F / F -F =1+(1/K[C] )
0
X
0
where, F , F , and F are the emission intensities of
lim
0
x
2
+
2 +
the probe in absence of Zn , at an intermediate Zn
concentration, and at a concentration of the complete
interaction with Zn respectively. K is the binding
Fig 15 Job’s plot for the determination of stoichiometry of [SALTM-
2
+
2+
ex
Zn ] complex in 100 % water at pH 7.4 (λ =300 nm, λem=450 nm)

J Fluoresc
Fig. 16 Relative emission
2+
intensities of SALTM-Zn
system in presence of various
cations in 100 % water at pH 7.4.
Black bars represent the emission
2+
intensity of the [SALTM-Zn ]
system and red bars show the
emission intensity of the
2+
[
SALTM-Zn ] system in
presence of 100 equiv. of different
2
+
2+
cations, viz. Cu (1), Mn (2),
2+
2+
2+
3+
Cd (3), Mg (4), Hg (5), Cr
2+
3+
2+
(
6), Ni (7), Al (8), Pb (9), +
3+ 2+ +
Fe (10), Co (11), K (12), Na
13), λex=300 nm, λem=450 nm
(
2
+
addition of Zn with the appearance of a new band at
3
Computational Studies
2
+
60 nm which gradually increases up to 40 equiv. Zn
with a clear isosbestic point at 385 nm, implying the
To rationalize the photo-physical properties of
SALTM and its Zn adduct, density functional the-
oretical (DFT) calculations have been performed
using 6-31++G (d, p) and LANL2DZ basis sets. No
significant difference between the results of two
2
+
2+
formation of SALTM- Zn adduct. The changes of ab-
2
+
sorbance have been recorded up to 800 μM Zn
Fig. 20). The linearity is observed up to 100 μM
(
2
+
2+
Zn , useful for determination of unknown Zn
concentration.
Fig. 18 Changes in the emission spectra of HNTM (20 μM) in HEPES
buffered solution (ethanol: water,1:1, v/v, pH 7.4) upon gradual addition
of Zn (0.10, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 25.0,
2
+
2+
Fig. 17 Binding constant of SALTM for Zn (Benesi-Hildebrand
method)
50.0, 100.0, 150,200, 250, 300, 350 and 400.0 μM) (λex=380 nm)

J Fluoresc
Fig. 19 Changes of absorbance
of SALTM (20 μM) upon
2+
addition of Zn (0.1, 0.2, 0.5,
1
9
2
8
.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0,
.0, 10.0, 25.0, 50.0, 100.0,
00.0, 400.0, 600.0 and
00.0 μM) in 100 % water at
pH 7.4
methods has been observed. However, for SALTM-
insight of emission properties of SALTM upon inter-
action with Zn . Absorbance spectrum of aqueous
solution of SALTM shows four bands centered at
405, 313, 276 and 251 nm.
2
+
2+
Zn adduct, LANL2DZ basis set is used. The opti-
2
+
mized geometries of SALTM and its Zn adduct are
shown in Fig. 21. TD-DFT studies provide deeper
Fig. 20 Variation of absorbance
of SALTM in 100 % water at
pH 7.4 (300 nm) as a function of
2+
externally added Zn (0.1–
00.0 μM)
8

J Fluoresc
Fig. 21 Energy optimized
structures of SALTM and its Zn
adduct
2+
Table 7 indicates that HOMO to LUMO (oscillator
strength, f = 0.1559) and HOMO-2 to LUMO (f =
.1398) are allowed transitions for SALTM (Fig. 22)
and 267 nm. Thus, theoretical studies are in line with
the blue shift of emission band of SALTM in presence
of Zn .
2
+
0
involving emissions at 284 nm and 251 nm. This is in
close agreement to experimental findings. Upon addi-
HeLa Cell Imaging
2
+
tion of Zn to the aqueous solution of SALTM, the
above mentioned bands disappear with the appearance
of two new bands at 360 nm and 269 nm. Computation-
2
+
Fresh media is supplemented with Zn and SALTM
accordingly and the cells are incubated for 2 h to
2
+
2+
al studies on [Zn - (SALTM-H)] complex predicts al-
facilitate Zn or SALTM uptake. After incubation,
lowable transition from HOMO −4 to LUMO (f=
cells are observed under an inverted fluorescence mi-
croscope (Dewinter, Italy) at different magnifications
with UV filter. SALTM is then added to Zn treated
0
.2407) and HOMO −2 to LUMO +1 (f=0.40401)
2
+
(
Fig. 23) (Table 8) with emission wavelengths at 354
Table 7 Electronic transitions observed from TDDFT studies of SALTM
Electronic transition
MOs involved
CI
Excitation energy/Wavelength,nm
4.1568 eV/298 nm
Oscillator strength (f)
0.0017
S
0
→S
1
HOMO-2→LUMO
HOMO-1→LUMO
−0.1083
0.6566
HOMO-1→LUMO+2
−0.1077
S
0
0
→S
2
3
HOMO-2→LUMO+1
HOMO→LUMO
0.1193
0.6546
4.3585 eV/284 nm
4.9407 eV/251 nm
0.1559
0.1398
S
→S
HOMO-3→LUMO
HOMO-2→LUMO
0.1449
0.6379
HOMO→LUMO+1
−0.13818

J Fluoresc
clearly indicates that significant numbers of in vitro
cells are alive under the experimental conditions
(
cell imaging requires maximum 2.5 h whereas
MTT assay have been performed after 48 h) in pres-
ence of the probe. The fluorescence cell imaging has
been performed using excitation wavelength,
3
70 nm.
Conclusion
2
+
Two new Zn
selective fluorescence probes, viz.
SALTM and HNTM have been reported. Both the
probes are structurally characterized by single crystal
X-ray analyses. SALTM functions in fully aqueous me-
dium in a ratiometric manner while HNTM acts as a
normal turn-on sensor in HEPES buffer medium. More-
over, SALTM may be easily and quickly synthesized in
a single step from readily available starting materials.
Fig. 22 Frontier molecular orbitals involved in the absorption spectrum
of SALTM
2
+
Zn assisted CHEF of SALTM is attributed to the in-
hibition of CH=N isomerization leading to enhanced
rigidity of the system. High water solubility of the
probe makes it a potential candidate for biological
cells and observed under microscope again. Images
are recorded with an attached CCD camera equipped
with Bio-Wizard 4.2 software (Fig. 24).
MTT assay has been performed to check the tox-
icity of the probe to the living cells. Figure 25
2
+
Zn monitoring in HeLa cancer cell. MTT assay sug-
gests non-harmful nature of the probe towards living
cell. The lowest LOD and binding constant of the probe
2
+
for Zn are quite satisfactory.
Fig. 23 Frontier molecular
orbitals involved for the
2+
absorption of [Zn - (SALTM-
H)] adduct

J Fluoresc
Table 8 Electronic transitions observed from TDDFT studies of [SALTM-Zn ] complex
2
+
Electronic transition
Composition
CI
Excitation energy/Wavelength,nm
3.4970 eV/354 nm
Oscillator strength (f)
0.0737
S
0
S
0
S
0
→S
1
8
HOMO→LUMO
HOMO→LUMO+1
0.65560
0.10203
→S
HOMO-3→LUMO+1
HOMO-2→LUMO+1
0.17930
0.40401
4.6279 eV/267 nm
4.7113 eV/263 nm
0.2153
0.2407
→S10
HOMO-4→LUMO
HOMO-3→LUMO
0.45499
0.28738
HOMO −5→LUMO+1
0.14883
Fig. 24 Fluorescence
microscope images of HeLa cells:
a and b are images after
2+
incubation with Zn (20 μM)
and SALTM (20 μM)
respectively. c Cells incubated
2+
with Zn (20 μM) followed by
addition of SALTM (20 μM,
λex=370 nm)
Fig. 25 MTTassay of HeLa cells
after treatment with SALTM at
different concentration for 48 h;
for 1, *p<0.05 significantly
different

J Fluoresc
2
+
Acknowledgments B. Kumari and S. Lohar are thankful to BU and
CSIR for fellowship. Assistance from CAS (UGC) is gratefully
acknowledged.
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2
4. Zhu J, Yuan H, Chan W, Lee AWM (2010) A colorimetric and
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