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S. Sinha et al. / Journal of Photochemistry and Photobiology A: Chemistry 277 (2014) 75–81
turn-on molecular probes which offer real-time analysis option for
in vivo studies and with high degree of selectivity for Zn2+ over
Na+, K+, Ca2+ and Mg2+ is an important and challenging area of
research.
but not with ligand and cells without Zn(NO3)2·6H2O treatment but
incubated with ligand were chosen as control.
2.5. Zinc imaging in gram seed sprouts
The present work demonstrates our recent studies on utiliza-
tion of TRIS-based molecular probe as Zn2+-specific fluorescence
bioimaging material. The in vitro zinc imaging potential of the
present probe was established using Bacillus thuringiensis cell line.
The imaging of zinc in a whole plant and in gram sprouts supported
the probe’s capability as bioimaging material for in vivo studies in
real-time.
Matured gram seeds were germinated at room temperature
using distilled water. The sprouted seeds were then treated with
Zn(NO3)2·6H2O solution for 2 h. After completion of zinc incuba-
tion, the seeds were washed several times with water to remove
surface sticking Zn(NO3)2·6H2O. Some of these incubated seeds
were then further incubated with probe L2 (0.5 mM) for 1 h. After
completion of incubation with probe L2, the seeds were thor-
oughly washed and then sectioned. Finally, the sections were
imaged using a fluorescence microscope. Sprout sections treated
with Zn(NO3)2·6H2O but not with probe L2 and sections without
Zn(NO3)2·6H2O treatment but incubated with probe L2 were used
as control.
2. Experimental
2.1. Materials
Chemicals and solvents were obtained from commercial sources
and used as received. Spectroscopic grade DMF was used to perform
all spectroscopy related experiments. The metal salts were used
in their nitrate form (except Hg2+ and Mn2+ which were used as
chloride salts). To obtain a fixed pH, 10 mM pH 7.0 HEPES buffer
was used as aqueous medium. Quantum yield was calculated using
quinine sulfate as reference.
3. Results and discussion
3.1. Synthesis
Probes L1/L2/L3 were synthesized using a simple one-pot con-
dition (Scheme 1). The products were obtained as yellow/red solids
and thoroughly characterized using spectroscopic tools such as FT-
IR, NMR and mass spectroscopy. The spectral data of L1 and L3 were
in agreement with the reported data [68,69].
2.2. Instruments
Absorption and emission spectra were recorded on Simadzu
UV-2450 and Cary Eclipse fluorescence spectrophotometers
respectively at ∼25 ◦C. All spectral studies were performed at 1 cm
quartz cell with 5 slit widths for both excitation and emission. FT-IR
spectra were recorded on a Perkin Elmer Spectrum 2 spectropho-
tometer. 1H and 13C NMR spectra were recorded on Jeol JNM ECX
400 MHz and Bruker Avance 300 MHz spectrometer in DMSO-d6.
The imaging system was comprised of an inverted fluorescence
microscope (Leica DM 1000 LED), digital compact camera (Leica
DFC 420C), and an image processor (Leica Application Suite v3.3.0).
The microscope was equipped with a mercury 50 W lamp.
3.2. UV–vis and fluorescence studies
To study the photo physical properties of ligands L1/L2/L3, in
the presence and absence of Zn2+, we started with UV–vis titra-
tion of L1. Three peaks were observed in the absorption spectra of
L1 (2 × 10−5 M) in DMF/Water (9:1) at 280, 317, and 409 nm (Fig.
S1). Addition of increasing amount of Zn2+ (0–16 × 10−5 M) resulted
in generation of a new absorption band centered at 364 nm. After
binding with Zn2+ the absorption peak at 409 nm almost dis-
appeared and a strong decrease in intensity in absorption band
centered at 317 nm was observed. Appearance of two isobestic
points at 330 and 398 nm clearly indicated the formation of L1-Zn2+
at 364 nm upon the addition of Zn2+ could be explained on the basis
of internal charge transfer (ICT) mechanism. The UV–vis spectra of
L2 showed three peaks at 285 nm, 346 nm and at 439 nm, while
L3 showed absorption peaks at 280 nm, 329 nm, and at 418 nm
(Fig. 1, S2). UV–vis titrations of L2/L3 (2 × 10−5 M) in a combina-
tion of DMF/Water (9:1) were performed with the gradual addition
of Zn2+ to check the effects of Zn2+ on the absorption of L2/L3 (Fig. 1,
2.3. Binding constant calculation
The equation that was used to calculate binding constant is
[G]tot = a/2K21(1 − a)2[H]tot + a[H]tot/2 [66,67], where [G]tot is total
concentration of guest (here Zn2+), [H]tot is the total concentration
of host, a = (A − Ao)/(Ainf − Ao) where A is the absorbance at a par-
ticular Zn2+ concentration, Ao and Ainf are the absorbances at zero
and infinite Zn2+ concentrations, respectively.
2.4. Cell culture and intracellular zinc imaging
B. thuringiensis (strain isolated in our laboratory as a bio-
pesticide agent for controlling looper pest of tea and identified on
the basis of 16S rDNA gene sequence homology) cells from expo-
nentially growing culture in Nutrient broth (pH 7.2, incubation
temperature 29 ◦C) were collected by centrifugation at 3000 rpm
for 5 min. After collection, these two types of cells were washed
twice by suspending them in 0.1 M HEPES buffer (pH 7.4) followed
by centrifugation in the same speed as above. The washed cells
were then treated separately with the zinc salt, Zn(NO3)2·6H2O
(0.05 mM) for 30 min. After completion of this incubation process
the cells were further washed with 0.1 M HEPES buffer (pH 7.4).
The washed cells were then further incubated with L2 (0.05 mM)
for another 30 min. Finally, and to minimize background fluores-
cence, the treated cells were washed further and then mounted on
grease free glass slide and observed under a Leica DM 1000 Fluores-
cence microscope with UV filter. Cells treated with Zn(NO3)2·6H2O
OH
HO
OH
OH
HO
H2N
OH
CHO
EtOH
OH
N
Reflux, 3-4 h, 55-60%
R
R= H, OMe, Br
R
OH
OH
OH
HO
HO
OH
OH
OH
HO
OH
OH
OH
OH
N
N
N
Br
OMe
L1
L3
L2
Scheme 1. Syntheses of L1, L2 and L3.