ESIPT and CHEF based highly sensitive and selective ratiometric sensor for Al3+ with imaging in human blood cells

Article abstract of DOI:10.1039/c5nj01468a

Based on excited state intramolecular proton transfer (ESIPT) and chelation enhanced fluorescence (CHEF) mechanisms, a new fluorescence ratiometric probe for Al³⁺ was designed and synthesized, and its structure was confirmed through single crys

Full text of DOI:10.1039/c5nj01468a

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ESIPT and CHEF based highly sensitive and selective ratiometric
sensor for Al3+ with imaging in human blood cell
Sangita Das,a Shyamaprosad Goswami,*a Krishnendu Aich, a Kakali Ghoshal, b Ching Kheng Quah,c
Maitree Bhattacharyya b and Hoong-Kun Fun c,d
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Based on excited state intramolecular proton transfer (ESIPT) and chelation induced enhanced fluorescence (CHEF)
machanisms, a new fluorescence ratiometric probe for Al3+ was designed and synthesized and its structure was confirmed
through single crystal X-ray study. This probe is capable to show excited state intramolecular proton transfer through two
different pathways. Introduction of Al3+ in the mixed aqueous solution of the probe exhibits abrupt change in the
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photophysical properties. A ratiometric emission profile was noticed in presence of Al3+
. Interestingly, the presence of
other metal ions (specially trivalent ions e. g. Fe3+, Cr3+, Ga3+ and In3+ ) do not perturb the fluorescence intensity of the
probe (except Cu2+ and Pb2+, slight changes were noticed). This indicates that the probe shows high affinity towards Al3+.
The ratiometric sensing phenomenon may be explained as the presence of two different mechanism namely excited state
intramolecular proton transfer and chelation induced enhanced fluorescence, showed by the probe in presence of Al3+ in
the excited state. The complexation of the probe with Al3+ inhibits excited state intramolecular proton transfer while the
chelation induced enhanced fluorescence mechanism come to play. The probe was efficient to detect the cellular uptake
of Al3+, which is demonstrated here with human blood-cell imaging. Moreover the detection limit was found to be 6.72 ×
10-8 M.
organs and tissues before being excreted through urine.
According to WHO the tolerable limit of weekly intake of Al3+
by human beings is around 7 mg Kg-1 body weight.8 Sensitive
Introduction
Aluminium is well known for its wide use in daily life as it is
found abundantly in nature and the third most prevalent metal
in the earth's crust. Increasing proofs recommend that
aluminium has inconsiderable toxicity in biological system.1
Aluminium compounds acting as paradox in our day to day life
as they are broadly used commercially in different industries
like paper industry, textile industry, alimentary industry4,5
etc, but very toxic to human life, flora, and fauna. 6 In addition,
frequent use of aluminium foil, vessels and cookie sheets in
bio-imaging of Al3+ in the cell is
a requirement for
understanding the fundamental mechanism about how
aluminium ions cause aluminium-induced human diseases.
Thus there is a great need to develop a device, enable to
detect a trace level of Al3+ in environmental and biological
samples and to control the concentration levels in the
biosphere and minimize direct effects on human health.
Molecular sensors are highly valuable tools for selective
recognition of chemical and biological species. There are many
interesting sensors reported for the detection of various
analytes involving intramolecularly hydrogen bonded property,
2
3
our daily life results in
a
reasonably increase of Al3+
concentration in food. Its excessive intake leads to severe
health problems such as gastrointestinal ailments, Alzheimer’s
disease, colic, rickets, osteoporosis, anaemia, and headache
etc.7 It can also damage the central nervous system in humans.
Aluminium ions may stay for a prolonged time in various
which exhibits excited state intramolecular proton transfer
9, 12
(ESIPT)
through tautomerization. In the past few years,
many fluorescence chemosensors have been reported for the
detection of Al3+.10 However, most of them are typical off-on
or on-off type. In this regard the sensors able to detect Al3+
through
a
ratiometric signal are seldom reported.11
Ratiometric fluorescent sensors usually allow to measure the
emission intensity at two different wavelengths which is
beneficial for the evaluation of analytes more accurately
because of its independency on the local probe concentration.
In our previous works,12 we have reported the ESIPT
mechanism by using HBT (hydroxybenzothiazole) derivative,
where the phenolic –OH plays the tautomerism with –N atom
of benzothiazole moiety. In continuation of our work, 13 here,
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we report the synthesis and photophysical properties of a system (S1/N1/C1-C7, r.m.s. deviation = 0.005 Å) forms
probe (HBTP) (pyridine conjugated hydroxybenzothiazole) for dihedral angles of 8.58(16) and 7.64(17)° with the benzene
the selective detection of Al3+ based on two different (C8-C13) and pyridine (N4/C16-C20) rings, indicating they are
mechanisms (ESIPT and CHEF).
almost coplanar to each other. The dihedral angle between
From single crystal X-ray study, it is confirmed that the probe benzene and pyridine rings is 1.49(19)°. The crystal structure
HBTP shows an intramolecular hydrogen bond with phenolic – features for intramolecular O—H···N hydrogen bond producing
OH and hydrazide linker. Consequently in the excited state the an S(6) ring motif, which is assumed to contribute to the
probe may exhibit two diffrent ESIPT by using phenolic –OH & stability of the molecule. In the crystal packing (Figure S10,
hydrazide linker and phenolic –OH & –N atom of benzothiazole ESI), the HBTP molecules are connected to the solvent
system. Introduction of Al3+ forms a stable complex with the molecule via intermolecular O3—H1O3···O4, N3—H1N3···O4
probe and inhibits the ESIPT. Consequently, the chelated and C14—H14A···S2 (Table S2, ESI) hydrogen bonds and are
system exhibits a brilliant cyan emission which may be due to stacked along [010], result in the formation of R1 (10) and
2
2
chelation induced enhanced fluorescence (CHEF). As per our R2 (7) ring motifs. The molecular packing is stabilized by weak
knowledge, ESIPT and CHEF blended mechanism for the C—H···π interactions involving the centroid of the C1-C6
selective detection of Al3+ has rarely been reported. Possible benzene ring.
practical utilization of HBTP as intracellular sensor of Al3+ was
also examined by human blood cell imaging using confocal
fluorescence microscope.
Results and discussion
The synthetic scheme of the probe is shown in Scheme 1.
Compound 6-(hydroxymethyl)picolinehydrazide (
by adopting the literature procedure, previously reported by
us.14 As depicted in Scheme 1, compound
3-
(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde) was
treated with compound to afford the final probe (HBTP) as
A) is prepared
3
(
A
Figure. 1 The molecular structure of the probe (HBTP), showing 50% probability
displacement ellipsoids for non-H atoms and the atom-numbering scheme.
Hydrogen bonds are represented by dashed lines.
light yellow solid in 81% yield. The structure of HBTP was
confirmed by 1H NMR, 13C NMR, HRMS and single crystal X-ray
study (ESI, Fig. S1-S9).
Sensing of Al3+ by UV-vis method
To examine the selectivity of HBTP, UV-vis and fluorescence
titration experiments were performed using common
interfering analyts (Na+, K+, Ca2+, Ni2+, Mn2+, Zn2+, Cd2+, Ga3+,
Cu2+, Fe3+, Cr3+, Pb2+, In3+, Hg2+ and Al3+ in H2O/MeOH (9/1, v/v,
10 mM PBS buffer, pH = 7.3, 250C) solution. The changes in
absorption spectra during the Al3+ titration have shown in Fig.
2a. HBTP (10 μM) itself showed two peaks at 270 nm and 370
nm in the absorbance profile.
+
+
HBTPandNa , K ,
(a)
(b)
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
2+ 2+
2+ 2+
Ca , Ni , Mn , Zn
,
2+
3+ 3+ 3+
Cd , Ga , Fe , Cr
,
2+ 3+ 2+
Scheme 1. Reagents and conditions: (i) 2-aminothiophenol, p-TSA, reflux, 6 h; (ii)
MnO2, CHCl3, reflux, 12 h; (iii) 6-(hydroxymethyl)picolinohydrazide, EtOH, reflux,
3 h.
Pb , In , Hg
Crystallographic study of the probe (HBTP)
3+
Al
2+
Cu
X-ray analysis reveals that HBTP crystallizes in orthorhombic
system with space group Pbca with a = 23.177 (5) Å, b = 8.5606
(17) Å, c = 23.924 (5) Å, Z = 8 and V = 4746.9 (17) Å3. A
summary of crystal data and parameters for structure
refinement details are given in Table S1 (ESI) whereas the
hydrogen-bond geometry can be found in Table S2 (ESI). The
asymmetric unit (Fig. 1) comprises a HBTP and a dimethyl
sulfoxide solvent molecule. The C12—C14—N2—N3 torsion
angle of 179.9(3)◦ involving the N2 = C14 (bond length = 1.283
(5) Å) double bond supports the (E)-configuration of the
structure. In HBTP, the mean plane of benzo[d]thiazole ring
250 300 350 400 450 500 550 250 300 350 400 450 500 550
Wavelength (nm)
Figure. 2 (a) Absorption spectra of HBTP (10 µM) upon titration with Al3+ (0 to 2
equivalents) in CH3OH/H2O (1/9, v/v, pH = 7.3, 250C) solution. (b) Changes of
absorption spectra of HBTP (10 µM) upon addition of different cations (5
equivalents).
Wavelength(nm)
Upon addition of increasing concentrations of Al3+ (0-2 equiv.)
the first two bands around 270 nm and 370 nm got decreased
regularly and a new peak at 440 nm gradually increases. Only
Al3+ has been successful to perturb the absorption signal of
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HBTP but other analytes cannot affect the absorption profile in In case of Cu2+ and Pb2+ a slight quench of fluorescence
a significant extent (Cu2+ showed slight interference) (Fig. 2b). intensity at 565 nm was noticed with no interference at 480
It has been observed that the absorbance intensity at 440 nm nm. Figure S15 (ESI) shows a comparative view of emission
exhibits
a
good linear relationship with added Al3+ intensity of the probe HBTP after adding 3.0 equiv. of each of
concentration from 0 to 10 µM, after that plateau was the guest analytes in presence of Al3+. This indicates with
reached, indicates the saturation takes place. In the presence background of most selected coexistent metal ions does not
of Al3+ ion, the color of the solution of HBTP changes from interfere the sensing of HBTP for Al3+. A comparison of the
colourless to light yellow. The association constant for the Al3+ present probe with recently reported probes for Al3+ have
complexation with HBTP was estimated to be 1.24 × 105 M-1 been outlined in Table S4 (ESI).
15
from Benesi-Hildebrand plot using the data obtained from We have performed the fluorescence titration of the probe
UV-vis titration (Fig. S11, ESI).
with Al3+ in different solvent systems like DMSO only,
ethanol/water (1/9) and DMSO/water (2/98) and results are
given in the ESI (Fig. S20 – S22). The results were almost similar
to that of methanol-water system (Fig. 3a).
Sensing of Al3+ by fluorescence method
The metal binding ability of the probe (HBTP) was evaluated by
fluorescence spectral analysis. Herein we have elaborated the
fluorogenic signalling behaviour of HBTP (10 μM) in
CH3OH/H2O (1/ 9, v/v, and pH = 7.3, 10 mM PBS buffer, 250C)
solution towards the detection of Al3+. Fig. 3a shows the
florescence spectrum of the probe (HBTP) in the presence of 2
equiv. of Al3+. Free probe displayed two broad bands at 435
nm and 565 nm. Upon incremental addition of Al3+ ion (0-2
equiv.) the peak at 565 nm decreased whereas a new band at
480 nm increases rapidly. Upon addition of almost two
equivalents of Al3+, the intensity at 480 nm increased by
almost 25 times than that of the probe itself. Consequently, a
prominent fluorescence colour change was observed from red
to cyan (Fig. 3a, inset). The changes in the fluorescence spectra
stopped when the amount of added Al3+ reached 1.2
equivalents. A good linear relationship was observed between
Sensing mechanism of the probe with Al3+
The two-way ESIPT of the free probe (HBTP) was given in
scheme 2, where the equilibrium 1 is predominant over
equilibrium 2. From single crystal X-ray study, it is confirmed
the existence of intramolecular hydrogen bond between
phenolic –OH and hydrazide linker, this explains the
predominance of equilibrium 1 in the free probe. The change
in colour of fluorescence after introduction of Al3+ was
observed mainly due to the inhibition of ESIPT. As from the
HSAB concept Al3+ being a hard acid loves to bind with hard
centers, like oxygen atom of the ethanol moiety, nitrogen
atom of the pyridyl moiety, hydrazide nitrogen atom and
oxygen of phenolic –OH group. The guest analyte Al3+ prone to
bind in this site, thus ESIPT stops and CHEF plays the key role
resulting cyan emission. Because of the isomerization in the
imine segment (-C=N), the probe may take any conformation,
either ‘E’ or ‘Z’. Consequently, the probe is more flexible in this
situation. But after addition of Al3+, restriction in the flexibility
is being employed and the complex become rigid and this may
also plays the key role for the enhancement in fluorescence.
the ratio of fluorescence intensities (I480/I565
)
and
concentration of added Al3+ (0 – 12 μM, Fig. S12, ESI). The
detection limit of the probe for Al3+ was determined from the
fluorescence spectral change, and it was found to be 6.72 × 10-
8
M, using the equation DL = K × Sb1/S, where K = 3, Sb1 is the
standard deviation of the blank solution and S is the slope of
the calibration curve16 (Fig. S12, ESI). Notably, addition of
other co-existing analytes, even in excess amount, caused
insignificant change in the emission intensity of the probe (Fig.
3b). Especially no other trivalent ions (e.g. Fe3+, Cr3+, Ga3+ and
In3+) can cause a considerable change in the fluorescence
profile of the probe (Fig. S23, ESI).
(a)
3x106
(b)
3+
Al
3x106
2x106
+
+
,
HBTP and Na , K
2+ 2+ 2+
2x106
2+
,
Ca , Ni , Mn , Zn
2+ 3+ 3+ 3+
Cd , Ga , Fe , Cr
3+ 2+
,
In , Hg
1x106
0
1x106
0
2+
Pb
2+
Cu
Scheme 2: Probable two-way ESIPT of the probe HBTP
400 450 500 550 600 650400 450 500 550 600 650
Wavelength(nm)
The complexation of the probe with Al3+ (Scheme 3) was
1
confirmed through H NMR titration study (Fig. S16, ESI). The
Wavelength (nm)
1H NMR titration study clear the unavailability of –OH proton
in HBTP-Al3+ (the peak of phenolic –OH at δ = 13.28 ppm
Figure. 3 (a) Fluorescence spectra of HBTP (10 µM) upon titration with Al3+ (0 to
2 equivalents) in CH3OH/H2O (1/9, v/v, pH = 7.3, 250C) solution. Inset: Emission
colour change of HBTP upon addition of 2 equivalents of Al3+ after illumination
under UV light. (b) Emission spectra of HBTP (10 µM) upon addition of different
metal ions (5 equivalents). λex = 380 nm.
gradually vanished after addition of
1
equivalent Al3+),
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indicates that the phenolic proton may take part in assay was also performed with the probe in presence and
complexation with Al3+.
absence of Al3+; it was given in figure S19 (ESI). This
experiment shows that HBTP is non-toxic to living cells. Fig.
4a depicts the bioimaging of human PBMCs by HBTP when
there is no added Al3+ from outside. Here cells have shown
significant red fluorescence and very little or no green
fluorescence. Fig. 4d surprisingly shows a far greater green
intensity indicating the interactions between HBTP and Al3+.
Average fluorescence intensity has been calculated by
Olympus Fluoview FV 1000 software and results have been
given in Table S3 (ESI). The green/red ratio is significantly
lower in samples without added Al3+ (0.046) where its value is
far higher (6.2) when externally Al3+ is being added. The
green/red florescence ratios implicate the shifting of green
fluorescence from red fluorescence by externally added Al3+.
Thus, HBTP has been proven to be a successful probe for
bioimaging.
Scheme 3. Probable binding mode of the probe with Al3+
Now no peak at δ = 5.56 ppm, also indicates the lack of
aliphatic –OH proton of the pyridine ring, this observation
showed that Al3+ chose to bind with the hard aliphatic oxygen
atom, confirm the complexation of Al3+ using this binding sight.
1
The H NMR titration study not only established the fact that
Al3+ plays as the key to lock ESIPT and favours CHEF but also
explains the exact binding site. The HRMS spectra of HBTP-Al3+
showed a peak at 479.0174 which may be for [M+Al3++2H2O–
2H+]+ (Fig. S17, ESI, Scheme 3). The IR spectra were recorded
for HBTP and HBTP-Al3+ complex which were given in Fig. S24
(ESI) also supported the complex formation. Depending on the
above findings we may conclude the probable complex
structure of HBTP-Al3+ and is given in Scheme 3.
The Job’s plot analysis was performed in order to establish the
exact stoichiometry of the complexation. In this experiment
we have recorded the change in emission at 480 nm against
molar fractions of HBTP and Al3+ under the conditions of an
invariant total concentration. The maximum point appears at
mole fraction of 0.5, which corresponds to a 1:1 (host–guest)
complex formation of HBTP and Al3+ (Fig S14, ESI).
Figure. 4 Confocal fluorescence images (60X) of human PBMCs treated with 10
µ
M HBTP (a & b) and then the same was treated with 10 µ
M Al3+ solution (d & e)
pH dependency of the probe and its complex with Al3+
respectively. Images were taken from (a & d) yellow channel (540 nm – 570 nm)
and (b & e) green channel (480 nm – 530 nm). λex = 400 nm. Larger cells are
macrophages in PBMC populations. C & f are the bright-field images of the cells
with probe before and after addition of Al3+ respectively.
In order to investigate the response of the probe towards
different pH, we have recorded the fluorescence spectra of
HBTP at different pH solutions (Fig. S18a, ESI). It was observed
that HBTP was almost nonresponsive within pH range 3-9. But
with increasing pH (10-12), a slight enhancement was noticed
at 480 nm. Now from the above experiment, it was concluded
that our probe is stable in biological pH, so that intracellular
experiment can be performed by it. The pH sensitivity of HBTP
in presence of Al3+ was also studied. The detection of Al3+ was
not affected within the pH range from pH = 1.2 to pH = 9, but
with increasing pH (˃ 10) the detecꢀon of Al3+ was a little bit
hampered. The pH study revealed that the probe HBTP can
detect Al3+ with no such interference in acidic, near neutral
and moderate basic pH conditions (Fig. S18b, ESI).
Conclusions
In summary, we report here the design, synthesis and cation
sensing property of a new hydroxybenzothiazole-pyridine
based probe. It showed highly selective and sensitive response
to Al3+ over other competing metal cations in MeOH/H2O (1/9,
v/v, pH = 7.3, 10 mM PBS buffer, 250C) media. A large
enhancement of ratiometric emission intensity was observed
after introduction of Al3+. The detection limit was found to be
10-8 M level, which indicates our probe (HBTP) is a highly
efficient sensor of Al3+ in mixed aqueous media. The
introduction of Al3+ in the solution of the probe (HBTP) stops
excited state intramolecular proton transfer and ‘turns on’ the
chelation induced enhanced fluorescence mechanism. Other
cations do not hamper the detection Al3+ in a significant
extent. The probe can also be used in human blood cell
imaging.
Live-cells imaging and cytotoxic effect of the probe and its Al3+
complex
HBTP is particularly useful for imaging of biological samples for
its permeability as well as stability in neutral pH. This
fluorescent probe is easily taken up by the cells without
causing any damages such as lysis or swelling. Cell viability
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aminothiophenol (0.45 g, 3.6 mmol) was added with
continuous stirring under the N2-atmosphere. A catalytic
amount of p-TSA (10 mg) was added to it and the reaction
mixture was refluxed for 6 hours. After that, the solvents were
evaporated under reduced pressure and the crude solid was
purified on a silica gel column chromatography using 5-10%
ethyl acetate in petroleum ether (v/v) as the eluant. The
product was obtained as light yellow solid. Yield = (0.65 g)
80%.
1H NMR (500 MHz, CDCl3): δ 2.36 (s, 3H), 4.80 (s, 2H), 7.22 (s,
1H), 7.43 (m, 2H), 7.51 (t, J = 7.7 Hz, 1H), 7.91 (d, J = 8 Hz, 1H),
7.98 (d, J = 8 Hz, 1H).
HRMS (ESI, positive): calcd. for C15H13NNaO2S [M + Na]+ (m/z):
294.0565; found: 294.0566.
Experimental
General
The materials used for this study were obtained from Sigma-
Aldrich and used without further purification. Merck 60 F254
plates with a thickness of 0.25 mm were used for thin layer
chromatography (TLC). 1H and 13C NMR spectra were recorded
on Brucker 400 MHz and 100 MHz instruments and mentioned
in the figure captions of the NMR spectra. CDCl3 or DMSO-d6
were used as solvent for NMR spectra using TMS as an internal
standard. UV-vis spectra were recorded on a JASCO V-630
spectrometer. Fluorescence spectra were recorded on PTI
(Photon Technology International) fluorescence spectrometer.
For the titration experiment we used the cations (metal ions)
viz. [Na+, K+, Ca2+, Ni2+, Mn2+, Zn2+, Cd2+, Cu2+, Fe3+, Mg2+, Cr3+
and Pb2+] as their chloride salts and Al3+ as its nitrate salt.
Synthesis of compound 3
MnO2 (0.26 g, 2.98 mmol) was added to a solution of 2 (0.4 g,
1.47 mmol) in chloroform (30 ml) under the N2-atmosphere.
Then the mixture was heated to reflux for 12 hours. The
reaction mixture was then passed through a celite bed (5 cm)
and to evaporate the solvent. The solid residues were
collected and purified on a silica gel column using ethyl acetate
in petroleum ether (1/4, v/v) as eluent. Compound 3 was
obtained as white solid. Yield = (0.28 g) 70%.
1H NMR (500 MHz, CDCl3): δ 2.44 (s, 3H), 7.47 (t, J = 7.5 Hz,
1H), 7.57 (t, J = 7.5 Hz, 1H), 7.72 (s, 1H), 7.95 (d, J = 8 Hz, 1H),
8.14 (d, J = 8 Hz, 2H), 10.42 (s, 1H).
HRMS (ESI, positive): calcd. for C15H11NNaO2S [M + Na]+ (m/z):
292.0408; found: 292.0405.
General method of UV-vis and fluorescence titration
UV-vis method
For UV-vis titrations, stock solution of the receptor (10 μM)
was prepared in [(CH3OH / water), 1/9, v/v] (at 25°C) using PBS
buffered solution. The solutions of the guest cations using
their chloride salts in the order of 1 × 10-5 M, were prepared in
deionized water using PBS buffer at pH = 7.3. Solutions of
various concentrations containing the sensor and increasing
concentrations of cations were prepared separately. The
spectra of these solutions were recorded by means of UV-vis
method.
Fluorescence method
Synthesis of the probe (HBTP)
For fluorescence titrations, stock solution of the sensor (10
μM) used was the same as that used for UV-vis titration. The
solutions of the guest cations using their chloride salts in the
order of 1 × 10–5 M, were prepared in deionised water.
Solutions of various concentrations containing sensor and
increasing concentrations of cations were prepared separately.
The spectra of these solutions were recorded by means of
fluorescence method.
6-(hydroxymethyl)picolinohydrazide (0.2 g, 1.19 mmol) was
added to a solution of 3 (0.32 g, 1.19 mmol) in ethanol (10 ml).
After addition, the reaction mixture was refluxed for 3 hours
and then cooled to room temperature. Yellowish-white
precipitate obtained was filtered and washed with cold
ethanol (2 × 2 ml). Yield = (0.4 g) 82%.
1H NMR (500 MHz, DMSO-d6): δ 2.39 (s, 3H), 4.74 (s, 2H), 5.56
(s, 1H), 7.46 (t, J = 7 Hz, 1H), 7.55 (t, J = 6.5 Hz, 2H), 7.72 (d, J =
7.5 Hz, 1H), 8.04 (m, 3H), 8.16 (m, 2H), 8.90 (s, 1H), 12.45 (s,
1H), 13.28 (s, 1H).
13C NMR (125 MHz, DMSO-d6): δ 19.8, 63.8, 119.4, 119.5,
120.9, 121.9, 122.2, 123.7, 125.0, 126.4, 128.6, 130.5, 132.9,
134.7, 138.3, 147.8, 149.2, 151.3, 154.0, 160.4, 161.0, 163.3
HRMS (ESI, positive): calcd. for C22H18N4NaO3S [M + Na]+
(m/z): 441.0997; found: 441.0996.
Determination of fluorescence quantum yield
To determine the quantum yields of HBTP and HBTP-Al3+, we
recorded their absorbance in methanol solution. The emission
spectra were recorded using the maximal excitation
wavelengths, and the integrated areas of the fluorescence-
corrected spectra were measured. The quantum yields were
then calculated by comparison with fluorescein (Φs = 0.97 in
basic ethanol) as reference using the following equation:
Φx = Φs × (Ix/Is) × (As/Ax) × (nx/ns)2
Method of crystallization
An amount of 20 mg of HBTP was dissolved in a small conical
in DCM/DMSO (1/1, v/v) solution. Then the conical was placed
gently in a cool place without any perturbation. After five days,
yellow crystals were obtained.
where, x & s indicate the unknown and standard solutions
respectively, Φ is the quantum yield, I is the integrated area
under the fluorescence spectra, A is the absorbance and n is
the refractive index of the solvent.
We calculated the quantum yields of HBTP and HBTP-Al3+ using
the above equation and the values are 0.087 and 0.46
respectively.
Synthesis of Al3+ complex (HBTP-Al3+) of receptor
The receptor, HBTP (50 mg) and Al(NO3)3 (14 mg) were mixed
together and dissolved in 5 ml of methanol. After reflux for 12
hours the reaction mixture was cooled to room temperature. A
reddish-yellow coloured precipitate appeared which was
filtered and dried in vacuum.
Synthetic method for the preparation of the probe
Synthesis of compound 2
To the suspension of
1 [2-hydroxy-3-(hydroxymethyl)-5-
HRMS (ESI, Positive): calcd. for C22H20AlN4O5S [M + Al3+ + 2H2O-
2H+]+ (m/z): 479.0953; found: 479.0174.
methylbenzaldehyde] (0.5 g, 3.01 mmol) in ethanol (10 ml), 2-
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DOI: 10.1039/C5NJ01468A
ARTICLE
Journal Name
Org. Biomol. Chem., 2011,
9
, 5523; (c) A. Banerjee, A.
Sahana, S. Das, S. Lohar, S. Guha, B. Sarkar, S. K.
Mukhopadhyay, A. K. Mukherjee and D. Das, Analyst, 2012,
137, 2166; (d) S. Goswami, K. Aich, S. Das, A. K. Das, D.
Sarkar, S. Panja, T. K. Mondal and S. Mukhopadhyay, Chem.
Commun., 2013, 49, 10739; (e) Y. Lu, S. Huang, Y. Liu, S. He,
L. Zhao and X. Zeng, Org. Lett., 2011, 13, 5274; (f) K. K.
Upadhyay, and A. Kumar, Org. Biomol. Chem., 2010, 8, 4892;
(g) S. Samanta, S. Goswami, Md. N. Hoque, A. Ramesh and G.
Das, Chem. Commun., 2014, 50, 11833; (h) S. Goswami, K.
Aich, A. K. Das, and S. Das, RSC Adv., 2013, 3, 2412.
Details of live-cell imaging
Materials Methods
3 ml of venous blood was obtained from volunteer donor (age
30 years) with his informed consent. Peripheral blood
mononuclear cells (PBMCs) were isolated by density gradient
centrifugation by histopaque-1077 obtained by SIGMA. PBMCs
were washed and suspended in PBS and were divided in two
sets. In one set, 10
source of Al3+. Another set was devoid of any externally added
µM Al(NO3)3 solution was added as a
11 (a) D. Maity and T. Gobindoraju, Chem. Commun., 2012, 48
,
Al3+. HBTP samples were prepared in PBS containing 0.5%
1039; (b) X. Sun, Y.-W. Wang and Y. Peng, Org. Lett., 2012,
14, 3420; (c) A. Sahana, A. Banerjee, S. Lohar, B. Sarkar, S. K.
Mukhopadhyay and D. Das, Inorg. Chem., 2013, 52, 3627.
12 (a) S. Goswami, S. Das, K. Aich, B. Pakhira, S. Panja, S. K.
Mukherjee and S. Sarkar, Org. Lett., 2013, 15, 5412; (b) S.
Goswami, A. Manna, S. Paul, A. K. Das, K. Aich and P. K.
Nandi, Chem. Commun., 2013, 49, 2912.
DMSO. Both the samples were incubated with 10 µM HBTP
solutions for 15 minutes at 370C. Cells were observed under
confocal fluorescence microscope (Olympus IX81 microscope)
with fluorescence emissions at 480 nm and 560 nm,
respectively.
13 (a) S. Goswami, K. Aich, S. Das, S. B. Roy, B. Pakhira and S.
Sarkar, RSC Adv
Aich, P. K. Nandi, K. Ghoshal, C. K. Quah, M.
Bhattacharyya, H.–K. Fun and H. A. Abdel-Aziz, RSC Adv
2014, , 24881; (c) S. Goswami, K. Aich, S. Das, A. K. Das, A.
Manna and S. Halder, Analyst, 2013, 138, 1903; (d) S.
Goswami, S. Das, K. Aich, D. Sarkar and T. K. Mondal,
Tetrahedron Lett., 2014, 55, 2695; (e) S. Goswami, S. Das and
K. Aich, Tetrahedron Lett., 2013, 54, 4620; (f) S. Goswami, K.
Aich, S. Das, D. Sarkar, C. D. Mukhopadhyay, D. Sarkar and
., 2014, 4, 14210; (b) S. Goswami, S. Das, K.
Acknowledgements
.
,
4
Authors thank CSIR and DST, Govt. of India for financial
supports. S.D, K.A and K.G acknowledge CSIR for providing
them fellowship. CKQ and HKF thank Universiti Sains Malaysia
for
Research
University
Individual
Grant
(No.
1001/PFIZIK/811278). The authors extend their appreciation to
The Deanship of Scientific Research at King Saud University for
the research group project No. RGP VPP-207.
T.K. Mondal, Dalton Trans., 2015, 44, 5763; (g) S.
Goswami, S. Das and K. Aich, RSC Adv., 2015, 5, 28996; (h) S.
Goswami, K. Aich, S. Das, B. Pakhira, K. Ghoshal, C. K. Quah,
M. Bhattacharyya, H.-K. Fun and S. Sarkar, Chem. Asian J.,
2015, 10, 694; (i) S. Goswami, S. Das, K. Aich, D. Sarkar and
T.K. Mondal, Tetrahedron Lett., 2014, 55, 2695; (j) K. Aich, S.
Goswami, S. Das, C. D. Mukhopadhyay, C. K. Quah, and H.-K.
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6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5NJ01468A
Graphical abstract:
We have synthesised a probe for fluorescence ratiometric detection of Al3+. The probe can image Al3+
in living cells.