Dual channel selective fluorescent detection of Al3+ and PPi in mixed aqueous media: DFT studies and cell imaging applications

Article abstract of DOI:10.1039/c5ra08024j

A new, easily synthesizable chemosensor, DFC-EN-p-Ph-NO₂, derived by the Schiff base condensation between 2,6-diformyl-p-cresol and N-(4-nitrophenyl)ethylenediamine, with potential N₄O donor atoms was found to act as a dual channel (colori- and fluorimetric) sensor towards Al³⁺ and PPi emitting at 486 nm (blue region) and 534 nm (green region), respectively in MeOH-H₂O (8 : 2, v/v) at pH 7.2 (10 mM HEPES buffer), μ = 0.05 M (LiCl), temperature 25 °C. The binding stoichiometries and formation constants of the sensor towards both Al³⁺ and PPi were determined by the combined UV-Vis and fluorescence titrations and Job's method, and confirmed by MS (m/z) studies. The corresponding detection limits as calculated by the 3σ method are: 7.55 μM and 3.34 μM. The most interesting part of this study is that on addition of 230 μM PPi to an ensemble of DFC-EN-p-Ph-NO₂-Al³⁺ (20 μM Ligand and 380 μM Al³⁺) the fluorescence is totally quenched but on further addition of PPi a new emission peak appears at 534 nm. All biologically relevant metal ions and toxic heavy metals did not interfere with the detection of Al³⁺ ion. Its bio-compatibility with respect to its good solubility in mixed organo-aqueous media (MeOH-H₂O) and cell permeability with no or negligible cytotoxicity provide good opportunities towards in vitro cell imaging studies of these ions. In particular, the fluorescent detection of PPi was not interfered by the presence of 400 μM of ATP or Pi although most reported PPi sensors that work in aqueous solution displayed cross-sensitivities toward ATP or Pi. The obvious excellent sensing capability of DFC-EN-p-Ph-NO₂ towards PPi and Al³⁺ was further scrutinized in HCT116 cell lines without much cytotoxicity. The modes of 1 : 1 binding of DFC-EN-p-Ph-NO₂ towards Al³⁺ and PPi were delineated by DFT calculations.

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Dual channel selective fluorescent detection of Al3+ and PPi
in mixed aqueous media: DFT studies and cell imaging
applications.
Cite this: DOI:
10.1039/x0xx00000x
Rabiul Alama, Tarun Mistria, Rahul Bhowmicka, Atul Katarkarb, Keya Chaudhurib and Mahammad Alia*
Received 00th January 2015,
Accepted 00th January 2015
DOI: 10.1039/x0xx00000x
Abstract
www.rsc.org/
A new, easily synthesizable chemosensor, DFC-EN-p-Ph-NO2, derived by the Schiff base condensation
between 2,6ꢀdiformylꢀpꢀcresol and Nꢀ(4ꢀnitrophenyl)ethylenediamie, with potential N4O donor atoms was
found to act as a double channels (coloriꢀ and fluorimetric) dual sensors towards Al3+ and PPi emitting at
486 nm (blue region) and 534 nm (green region), respectively in MeOHꢀH2O (8:2, v/v) at pH 7.2 (10 mM
HEPES buffer), ꢁ = 0.05 M (LiCl), temperature 25 ºC. The binding stoichiometries and formation
constants of the sensor towards both Al3+ and PPi were determined by the combined UVꢀVis and
fluorescence titrations and Job’s method, and confirmed by MS (m/z) studies. The corresponding
detection limits as calculated by 3σ method are: 7.55 ꢂM and 3.34 ꢂM. The most interesting part of this
study is that on addition of 230 ꢁM PPi to an ensemble of DFC-EN-p-Ph-NO2 ̶
Al3+ (20 ꢁM Ligand and
380 ꢁM Al3+) the fluorescence is totally quenched but on further addition of PPi a new emission peak
appears at 534 nm. All biologically relevant metal ions and toxic heavy metals did not interfere with the
detection of Al3+ ion. Owing to its bioꢀcompatibility with respect to its good solubility in mixed organoꢀ
aqueous media (MeOH/H2O) and cell permeability with no or negligible cytotoxicity provide good
opportunity towards inꢀvitro cell imaging studies of these ions. In particular, the fluorescent detection of
PPi was not interfered by the presence of 400 ꢁM of ATP or Pi although the most reported PPi sensors
that work in aqueous solution displayed crossꢀsensitivities toward ATP or Pi The obvious excellent
sensing capability of DFC-EN-p-Ph-NO2 towards PPi and Al3+ were further scrutinized in HCT116 cell
lines without much cytotoxicity. The modes of 1:1 binding of DFC-EN-p-Ph-NO2 towards Al3+ and PPi
were delineated by DFT calculations.
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Physical measurements
Introduction
Pyrophosphate (P2O74ꢀ, PPi), a hydrolysis product of ATP and other
nucleotide triphosphates, is involved in various anabolic and
bioenergetics processes.1ꢀ3 Several cell signalling processes utilize
adenylatecyclase (guanylatecyclase) that catalyze the conversion of
ATP (GTP) to cAMP (cGMP) and PPi.4,5 PPi is also used as an
additive in food (E450), toothpaste, baking powder, and detergents.6
Thus, the selective detection of PPi is important for monitoring the
biological processes mediated by hydrolysis of nucleotide
triphosphate to release PPi.
Elemental analyses were carried out using a Perkin–Elmer 240
elemental analyzer. Infrared spectra (400–4000 cmꢀ1) were
recorded in liquid state on a Nickolet Magna IR 750 seriesꢀII
1
FTIR spectrometer. HꢀNMR spectra were recorded in DMSOꢀ
d6 on
a Bruker 300 MHz NMR spectrometer using
tetramethylsilane (δ = 0) as an internal standard. ESIꢀMS+ (m/z)
of the ligand and Al3+ꢀcomplex were recorded on a Waters’
HRMS spectrometer (Model: XEVO G2QTof). UVꢀVis spectra
were recorded on an Agilent diodeꢀarray spectrophotometer
Since the first report on PPi fluorescent sensor by Czarnik7 this
becomes an interesting field of research by chemists and
biochemists.8ꢀ11 Most often, these sensors contain metal complexes
as the recognition units of phosphates. Zn(II) complexes are
frequently used in this context,12 but other metal ions such as
Cu(II),13 Cd(II),14 Mn(II),15 Eu(III),16 and Tb(III)17 have been employed
as well, however Al3+ bound PPi sensor is rare.18 Even though
various types of chemosensors that recognize PPi in water have
been reported,11,19ꢀ23 only a few of them had sufficient selectivity for
PPi over ATP or Pi in aqueous solutions.
(Model,
Agilent
8453).
Steadyꢀstate
fluorescence
PTI QMꢀ40
measurements were performed with
spectrofluorometer.
a
Solution Preparation for UV-Vis Absorption and fluorescence
studies
For both UVꢀVis and fuorescence titrations, stock solution of 1.0 x
10ꢀ3 M of the probe DFC-EN-p-Ph-NO2 was prepared in MeOH.
Similarly, another 1.0 x 10ꢀ3 M stock solution of Al(NO3)3.9H2O and
other metal ions were prepared in MeOHꢀH2O (8:2, v/v). A solution
of MeOHꢀH2O (8:2, v/v) containing 10.0 mM HEPES buffer was
prepared and pH was adjusted to 7.20 by using HCl and NaOH.
Ionic strength was maintained at 20 mM (NaCl) throughout the
measurements. 2.5 ml of this buffered solution was pipetted out into
a cuvette to which was added 20 ꢁM of the probe and metal ions
were added incrementally starting from 0 to 380 ꢁM in a regular
interval of volume and UVꢀVis and fluorescence spectra were
recorded for each solution.
Preparation of N-(2-aminoethyl)-4-nitrobenzenamine (1).
Nꢀ(2ꢀaminoethyl)ꢀ4ꢀnitrobenzenaminehas been prepared using
literature procedure.24
Scheme 1
In Scheme 1 we describe a turnꢀon Al3+ sensor which can
also functions as PPi sensor both in presence and absence of Al3+.
The most important aspects of this sensor are that it senses Al3+
selectively by fluorescence emission in blue region while PPi
sensing occurs in the green region.
Preparation of DFC-EN-p-Ph-NO2 (2).
2,6ꢀDiformylꢀpꢀcresol (DFC) was prepared by following a literature
procedure.25 2,6ꢀDiformylꢀpcresol(DFC ) (1.64 g, 10 mmol) was
dissolved in 25 ml EtOH under nitrogen atmosphere. To this
solution, Nꢀ(2ꢀaminoethyl)ꢀ4ꢀnitrobenzenamine (1) (1.81 g, 10 mmol)
was added and stirred at room temperature for 2 h, whereupon solid
precipitate formed was filtered and washed with diethyl ether and air
dried (Scheme 2). It was recrystallized from ethanol to get pure
product. Yield, 80%.CHN Analysis for C25H26N6O5: Calculated (%):
C, 61.22; H, 5.34; N, 17.13. Found (%): C, 61.21; H, 5.33; N, 17.14.
.1HꢀNMR (in DMSOꢀd6) (δ, ppm): 7.36 (s,2H (NH), 14.04 (s,1H),
2.23 (s, 3ꢀH), 7.48 (s, 2ꢀH), 8.57 (s, 2ꢀH) , 7.98 (d, J= 9.2, 4ꢀH), 6.70
(d, J=9.2,4H ), 3.78 (d, J=5.0,4H), 3.50 (d, J= 5.6, 4ꢀH) (please see
Materials and Methods
The starting materials such as pꢀchloro nitrobenzene (Sigma
Aldrich), ethylenediamine, diformylꢀpꢀcresol (prepared in this
laboratory) were used for the preparation of ligands. Al(NO3)3.9H2O
was used to prepare Al3+ꢀcomplex. Solvents like ethanol, diethyl
ether and methanol (Merck, India) were of reagent grade and dried
before use.
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1
Fig. S1 for HꢀNMR). 13CꢀNMR: (300 MHz, DMSOꢀd6) δppm: 20.27, 1:9 (v/v) in the culture medium) for 30 min at 37 °C. After incubation,
43.59, 58.68, 111.42, 121.30, 126.64, 127.02, 132.87, 136.21, cells were washed thrice with 1X PBS; cover slip containing HCT116
155.01, 159.70, 162.48 (Fig. S2).ESIꢀMS+ (m/z): 491.20(DFC-EN-p- cells were allowed to treated with 0ꢁM, 4ꢁM, 6ꢁM, 8ꢁM and 10ꢁM
Ph-NO2 + H+) [Fig. S3]. IR spectrum (in MeOH): ꢀOH (3499 cmꢀ1),ꢀ of Al3+ (Experimental SetꢀI), 0ꢁM, 4ꢁM, 6ꢁM, 8ꢁM and 10ꢁM of
C=N (1639 cmꢀ1), ꢀNH (3177 Cmꢀ1). (Fig.S4)
PPi (Experimental SetꢀII) and 0ꢁM, 4ꢁM, 6ꢁM, 8ꢁM and 10ꢁM of
Al3++PPi (Experimental SetꢀIII), incubated for 30 min at 37ºC,
washed twice with1X PBS and coverslip were mounted over the
slide. Bright field and fluorescence images of HCT116 cells were
taken by a fluorescence microscope (Leica DM3000, Germany) with
an objective lens of 40X magnification. HCT116 cells showed
excellent green fluorescence when DFC-EN-p-Ph-NO2 formed
intracellular complex with PPi and blue fluorescence with Al3+.
Synthesis of [Al(DFC-EN-p-Ph-NO2-H+)(H2O)(OH)(NO3)]Complex
(3).
Al(NO3)2.9H2O (0.375 g, 1 mmol) was dissolved in 10 ml
methanol.To this solution, DFC-EN-p-Ph-NO2 (0.350 g, 1.0 mmol.) in
3 mL MeOH was added. The colour of the solution changed to faint
yellow. The resulting mixture was stirred for 3 h at room
temperature. The volume of the solution was reduced to 5 ml under
reduced pressure and diethyl ether (10 mL) was added and kept at 0
°C for 12 h to afford complex 3 as microcrystals. Yield: 0.356 g
(~52%). CHN analyses for [Al(DFC-EN-p-Ph-NO2)(H2O)(OH)](NO3)]
C25H28N7O10Al (M.W.613.51), Calcd (%): C, 48.94; H,4.60; N, 15.98.
Found (%): C, 48.90; H 4.66; N, 15.97. UVꢀvis. (MeOH): λmax 370 nm
(Fig. 1).
Results and Discussion
Synthesis and Characterization of DFC-EN-p-Ph-NO2
DFCꢀENꢀpꢀPhꢀNO2 was prepared according to the synthetic routes
shown in Scheme 2 in 80 % yields.
To an ethanolic solution of 2,6ꢀDiformylꢀpcresol (DFC) under
nitrogen
atmosphere
was
added
Nꢀ(2ꢀaminoethyl)ꢀ4ꢀ
nitrobenzenamine (1) and stirred at room temperature for 2h,
whereupon solid precipitate formed was filtered, recrystallized from
ethanol to get the pure product. The probe was characterized by
1
various spectroscopic (IR, H and 13C NMR) and HRMS analyses. It
was further reacted with Al(NO3)2.9H2O to afford complex 3 as
microcrystals in 52% yield and characterized analogously. Several
attempts to get single crystals for Xꢀray diffraction studies were
failed.
Scheme 2
Cell culture
UV-Vis Absorption Studies
Human colon cancer (HCT116) cell line, were grown in DMEM
supplemented with 10% FBS and antibiotics (penicillinꢀ100 ꢁg/ml;
streptomycinꢀ50 ꢁg/ml). Cells were cultured at 37ºC in 95% air, 5%
CO2 incubator.
The electronic absorption properties of DFC-EN-p-Ph-NO2 (2) were
investigated in MeOH:H20 (8:2, v/v) at pH 7.2 in 10 mM HEPES
buffer at ꢁ = 0.05 M (LiCl). The absorption spectrum of the free
ligand consists of three absorption bands at 455, 388 and 276 nm
which are assigned as π–π*, n–π* and σ–π* transitions,
respectively.27 On gradual addition of Al3+ the absorbance of
compound 3 at 448 and 270 nm gradually decrease, while, the band
at 388 nm gradually shifted to 370 nm producing three well defined
isosbestic points at 375, 300 and 250 nm which clearly indicate the
transformation of free ligand to its metalꢀbound state.
Cell Cytotoxicity Assay
To determine % cell viability of DFC-EN-p-Ph-NO2, MTT assay was
performed.26 HCT116 cells (1×105 cells/well) were cultured in a 96ꢀ
well plate at 37°C, and exposed to varying concentrations of 1, 10,
20, 40, 60, 80 and 100 ꢂM of DFC-EN-p-Ph-NO2 for 12 h. 20 ꢂl of
MTT solution [5 mg/ml 1X phosphateꢀbuffered saline (PBS)] was
added to each well of a 96ꢀwell culture plate and again incubated
continuously at 370C for a period of 4 h. All media were removed
from wells and 150 ꢁl of DMSO was added to each well and
absorbance was measured at 550 nm (EMax Precision MicroPlate
Reader, Molecular Devices, USA). All experiments were performed
in triplicate and the relative cell viability (%) was expressed as a
percentage relative to the untreated control cells.
y = (a+b*c*xn)/(1+c*xn) (1)
When we plot absorbance as a function of [Al3+] a nonꢀlinear curve
of decreasing slope was obtained which can be easily solved by
using eqn (1),28 where a and b are the absorbances in the absence
and presence of excess metal ions, respectively, c (= K) is the
formation constant and n is the stoichiometry of the reactions. The
nonꢀlinear leastꢀsquares curveꢀfit of the absorption titration data
(Fig.1 b) as a function of [Al3+] gives: c = K = (2.26 ± 1.00) x 104 Mꢀ1,
n = 0.93 ± 0.04. The 1:1 mole ratio for LꢀM binding was further
determined by JOB’s method (Fig.S5) and ESIꢀMS+(m/z)(Fig. S3a)
studies.
Cell Imaging Study
HCT116 Cells were cultured and incubated in 35 x10 mm culture
dish over coverslip and allowed to incubate with 10ꢂm DFC-EN-p-
Ph-NO2 (prepared by dissolving in a mixed solvent (DMSO: water =
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As the reaction between the probe and Al3+ was found to be slow we the metal ion. Figure 3 displays a time resolved spectra for the
tried to evaluate the rate constants for this reaction under pseudoꢀ reaction between DFC-EN-p-Ph-NO2 and Al3+. Each spectrum was
first order conditions taking the probe as a minor component [DFC- taken at an interval of 50 seconds for initial 10 spectra and then it
EN-p-Ph-NO2] = 20 ꢁM and [Al3+] in large excess (20ꢀ200 ꢁM). In was recorded at an interval of 100 seconds. As the reaction between
each concentration of Al3+ the pseudoꢀfirstꢀorder rate constants were probe and Al3+ is relatively slow each spectrum for absorption
evaluated by fitting the kinetic traces to a firstꢀorder computerꢀfit titration was recorded after 2 hour of mixing.
program associated with the spectrophotometer. A plot of kobs vs.
[Al3+] yields
a nonꢀlinear curve of decreasing slope. Such
dependence could be best described by the following reaction
sequence (eqn 2).
(2)
The corresponding rate law could be derived as eqn (3)
Fig. 3.Time resolved spectra for the reaction between the probe
DFC-EN-p-Ph-NO2 and Al3+ in MeOHꢀH2O (8:2, v/v) at pH 7.2 (10
mM HEPES buffer), ꢁ = 0.05 M (LiCl), temperature 25 ºC. First
spectrum was taken immediately after mixing and subsequent
spectra were taken at a time interval of 60 seconds.
Fluorescence studies
Due to excited state intramolecular proton transfer (ESIPT) from the
phenolic –OH to azomethynꢀN atom along with the C=N
isomerization, the free ligand shows very weak fluorescence
behaviour at 520 nm. But on complexation with Al3+ both these
processes are effectively blocked and the probe becomes highly
fluorescencent through chelation enhanced fluorescence (CHEF)
effect resulting a large enhancement in the fluorescence intensity at
486 nm accompanied with 34 nm blue (Fig. 4). The mechanism is
shown in Scheme 1. A proton transfer and C=N isomerization might
also affect the “second” azomethin group in the molecule, even after
chelation of one Al3+ ion.
Fig. 1. (a) Absorption titration of with Al3+ in MeOHꢀH2O (8:2, v/v) in
HEPES buffer (10 mM) at pH 7.2; (b) Nonꢀ linear curve fitting.
Fig. 4. (a) Fluorescence titration of 20 ꢂMDFC-EN-p-Ph-NO2 in
MeOHꢀH2O (8:2, v/v) in HEPES buffer at pH 7.2 by the gradual
addition Al3+ with λex= 370 nm, λem = 486 nm Inset (b) nonꢀlinear
curveꢀfit of F.I vs. [Al3+].
Fig. 2. Nonꢀlinear curveꢀfitting of a plot of kobs vs. [Al3+] for the complexation
of Al3+ with DFC-EN-p-Ph-NO2 in MeOHꢀH2O (8:2, v/v) at pH 7.2 (10 mM
HEPES buffer), ꢁ = 0.05 M (LiCl), temperature 25ºC
Nonꢀlinear leastꢀsquares fitting of kobs as a function of [Al3+] gives k =
(2.6 ± 0.30) x 10ꢀ3 Mꢀ1sꢀ1 and Q = (1.10 ± 0.23) x 104 Mꢀ1. Here Q
signifies the initial innerꢀsphere association between the probe and
The fluorescence titrations were carried out in three ways. (a) In the
first step we kept ligand concentration fixed at 20 ꢁM and [Al3+]
varied between 0 and 380 ꢁM and Fluorescence intensity (FI) at
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486 nm were recorded (λex = 370 nm) at a fixed time interval of 30 The described sensor shows turn on fluorescent response to Al3+
min to allow the reaction to complete, as the reaction between the and PPi so the two analytes may interfere with each other during the
probe and Al3+ was found to be slower. When we plot FI as a detection. To short out how to avoid the possible interference from
function of [Al3+] we get a nonꢀlinear curve of decreasing slope which each other we have carried out two experiments. In one case, to 20
was solved by fitting the titration data to eqn 1 and the ꢂM probe, ~17 equivalents of Al3+ was added and then PPi was
corresponding evaluated parameters are: Kf = (5.29 ± 1.11) x 104 Mꢀ added incrementally. The emission band at 486 nm is abruptly
1
and n = 1.30 ± 0.02 which clearly signifies a 1:1 complexation quenched on addition of ~12 equivalents of PPi (Fig. S7) and a new
between DFC-EN-p-Ph-NO2 and Al3+.The detection limit as peak at 534 nm is started to grow on further addition of PPi to the
determined by 3σ method was found to be 7.55 ꢂM (Fig.S6)
solution mixture and becomes constant at [PPi ]= 700 ꢂM (Fig.S7).
In a second attempt (b) we have treated 20 ꢁM probe with 380 ꢁM In another experiment, to a 20 ꢂM probe, 700 ꢂM
(35 equivalents
)
[Al3+] and then added PPi gradually upto 230 ꢁM which showed a PPi was added and then Al3+ was added incrementally. It was
gradual decay of FI with added PPi. First spectrum was taken within observed that emission peak at 534 for DFCꢀenꢀpꢀPhꢀNO2─PPi
10 sec of mixing and subsequently each spectrum was recorded at a complex starts to diminish with the increase in concentration of Al3+,
time interval of 30 sec. This decrease in FI may be attributed to the becomes almost the same as that of in pure probe at 950 ꢂM of Al3+
displacement of Al3+ from Al3+–PPi complex and this arises due to a and then a peak at 484 nm for DFCꢀenꢀpꢀPhꢀNO2─Al3+ complex
stronger affinity of Al3+ towards PPi over the probe. On further stars to grow on further increasing the concentration of PPi and
addition of PPi to this mixture there was a gradual increase in FI at becomes constant at [Al3+] = 1225 ꢂM. These two experiments
534 nm which may be due to Hꢀbonding interaction between the clearly demonstrate the reversibility and selectivity of the probe
probe and PPi (Fig.S7) giving a rigid structure to the probe and towards Al3+ and PPi; one being higher in concentration over the
blocking both the ESIPT and isomerization across the C=N other (Fig.S8). The quantum yield value for free DFC-EN-p-Ph-NO2
(azomethyne) bond. Finally, (c) to confirm the proposition in (b) we and DFC-EN-p-Ph-NO2–Al3+, DFC-EN-p-Ph-NO2–PPi ensembles
have carried out this titration in the absence of Al3+ and indeed there are 0 .002, 0.011, 0.003 respectively.
is a gradual increase in FI at 534 nm on increasing the concentration
of PPi. A plot of FI at vs. PPi gives nonꢀlinear curve and solved by
fitting the titration data to eqn 1 and the evaluated parameters are:
Kf = (1.34 ± 0.81) x 103 Mꢀ1 and n = 0.94 ± 0.07.( Fig. 5). In order to
check the applicability of the probe at lower concentration we have
carried out experiments keeping probe concentration at 2 ꢁM and
then treated with increasing concentration of Al3+ and PPi separately
and we observed significant enhancement of FI at 484 and 534 nm
respectively, suggesting its applicability at lower concentration ( at
least at 2 ꢁM).
The selective sensing of analyte is an important criterion of a
successful sensor. In order to check the selectivity of DFC-EN-p-Ph-
NO2 towards Al3+ we have carried out steady state fluorescence
experiments with 20 ꢂM DFC-EN-p-Ph-NO2 and 5 equivalents of
different metal ions both in absence and presence of Al3+. It was
interesting to note that the detection of Al3+ was not perturbed by
biologically abundant metal ions like Na+, K+, Ca2+ etc and transition
metal ions, namely Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, as well as
heavy metal ions like Cd2+, Pb2+, and Hg2+(Fig. 6 ).
Fig. 5. (a) Fluorescence titration of 20 ꢂM DFC-EN-p-Ph-NO2 in MeOHꢀ
H2O (8:2, v/v) in HEPES buffer at pH 7.2 by the gradual addition PPi with λex
= 370 nm, λem = 534 nm Inset (b) nonꢀlinear curveꢀfit of F.I vs. [PPi] and the
evaluated parameters are: Kf = (1.34 ± 0.81) x 103 Mꢀ1 and n = 0.94 ± 0.07.
The strong solvatochromic behavior was observed for the free ligand
(DFC-EN-p-Ph-NO2), as well as its complexes with Al3+ (DFC-EN-p-
Ph-NO2-Al) and PPi (DFC-EN-p-Ph-NO2-PPi). In all cases there are
red shifts of absorption maximum on increasing the solvent polarity
which clearly indicates that there is charge separation in the excited
state and interact strongly with more polar solvents (Fig. S9). In
case of fluorescence emission spectra we observed solvent polarity
dependent blue shift for DFC-EN-p-Ph-NO2 and red shift for DFC-
EN-p-Ph-NO2-Al and DFC-EN-p-Ph-NO2-PPi. The blue shift in λem
of the free probe may be rationalized by considering low charge
separation in the excited state compared to the ground state,
possibly due to attainment of planar structure in the excited state. In
other cases normal behaviour (red shift) is apparent (Fig.8 and Fig.
With the addition of PPi, there is an enhancement in fluorescence
intensity (~5 fold) at 534 nm which is red shifted from 520 nm on
excitation at 370 nm). The detection limit is found to be 3.34 ꢂM
(Fig. S6a) indicating a high sensitivity of the ligand towards PPi. No
significant changes were observed in the emission spectra by the
addition of the other anions such as HPO42ꢀ, PO43−, ATP, AcO−, Cl−,
ꢀ
ꢀ
ꢀ
ꢀ
Br−, F−, SO42ꢀ, SCNꢀ and N3 , AsO4 , S2O4 , NO2 under similar
experimental conditions. The selectivity for PPi with 20 ꢂM of DFC-
EN-p-Ph-NO2 was plotted as a bar diagram in Fig.7. Interestingly,
anions other than PPi that contain the phosphate moiety do not S9).This is confirmed by a plot of Plot of Stokes shift ( ) vs
interfere in the sensing of PPi, except ATP where a slight FI solvent polarity parameter ET(30) in different solvents (Fig. S15ꢀ
enhancement was noted.
S17).29
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Fig. 6. (a) Fluorescence response of different metal ions towards DFC-EN-p-Ph-NO2(20 ꢂM); (b) Interference from other metal cations in a binary mixture:
L(DFC-EN-p-Ph-NO2 ) (20 ꢁM) + Al3+ (380 ꢁM) + Mn+ (5 equivalent than Al3+), where Mn+ = Ni2+, Fe3+, Cd2+, Mg2+ , Zn2+, Cr3+ , Co2+, Na+, Cu2+, Mn2+, Hg2+,
Pb2+ and Mn2+ . Mix = (Ni2+, Fe3+, Cd2+, Mg2+ , Zn2+, Cr3+ , Co2+, Na+, Cu2+, Mn2+, Hg2+, Pb2+, Mn2+) were present together with L and Al3+ (in MeOHꢀH2O
(8:2, v/v) in HEPES buffer at pH 7.2 , λex= 370 nm, λem = 486 nm). Each spectra was drawn after 15 mins.
Fig. 7.(a) Fluorescence response of different anions towards DFC-EN-p-Ph-NO2 (20 ꢂM); (b) Interference from other anions in a binary mixture: L(DFC-EN-
ꢀ
2ꢀ
ꢀ
2ꢀ
ꢀ
p-Ph-NO2) (10 ꢁM) + PPi (350 ꢁM) + Xnꢀ (5 equivalent than PPi ), where Xnꢀ = Brꢀ, Fꢀ, Clꢀ, SCNꢀ , N3 , ATP , HPO4 , NO2 , OAcꢀ, SO4 , S2O4 , AsO4ꢀ and
PPi . Mix = (Brꢀ, Fꢀ, Clꢀ, SCNꢀ , N3 , ATP , HPO4 , NO2 , OAcꢀ, SO4 , S2O4 , AsO4ꢀ) were present together with L and PPi (in MeOHꢀH2O (8:2, v/v) in HEPES
ꢀ
2ꢀ
ꢀ
2ꢀ
ꢀ
buffer at pH 7.2 , λex= 370 nm, λem = 534 nm).
Fig.8. Polarity dependent blue shift of λem of the ligand (nonꢀpolar to polar) and red shift of λem of the Alꢀcomplex (nonꢀpolar to polar)
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proton (f) signal from 7.36 to 7.17 in complex [Fig. 9(a)] is due to
electronic environment. In case of DFC-EN-p-Ph-NO2 PPi complex
̶
this OH proton signal is broadened and almost vanishes. The
azomethine proton (c) remains almost unchanged (7.36 to 7.25) or
slightly upꢀfield shifted due to nonꢀparticipation in Hꢀbonding to PPi
and shift of imine–NH proton (f) towards upꢀfield occurs due to Hꢀ
bonding [Fig. 9(b)].
We have also carried out DFT calculations on DFC-EN-p-Ph-
NO2, DFC-EN-p-Ph-NO2
̶ ̶ PPi to
Al3+ and DFC-EN-p-Ph-NO2
strengthen the coordination modes of the ligand towards Al3+ and
PPi and their optimized geometries are shown in Fig S10. The
TDDFT calculations on free ligand and its Al3+ complex were used to
generate their UVꢀVis spectra which showed excellent match in λmax
with experimental findings (Table S4). A blue shift in the absorption
spectrum of DFC-EN-p-Ph-NO2–Al3+ complex compared to free
ligand is also rationalized by comparing the HOMO–LUMO+1 gap
for these species which is slightly higher for the former (∆E =3.46eV
, λtheo=370) than the later (∆E=3.27eV, λtheo=388) ( Fig 10).
1
Fig. 9. (a) HꢀNMR shifts of free ligand and with addition of 1.5
equivalents of and Al3+ (b) PPi in DMSOꢀd6 recorded on a 300 MHz
Bruker 50 NMR spectrometer.
The coordination mode of DFC-EN-p-Ph-NO2 towards Al3+ was
supported by 1HꢀNMR studies (Fig. 9) which clearly showed a down
field shift of azomethine protons (c) in DFC-EN-p-Ph-NO2 –Al3+
complex from 8.57 of the free ligand to 9.42, 10.17 in the complex.
[Fig. 9(a)] The larger shift and splitting into two peaks are due to
strong binding of one azomethine N to Al3+ while other decoupled
from Hꢀbonding with phenolic –OH proton (Table S1). In the free
ligand there is intramolecular Hꢀbonding between azomethyneꢀN
and –OH proton (i) resulting a downfield shift of ꢀOH proton signal to
14.04 ppm and appears as a broad signal, which vanishes
completely on complexation with Al3+. The upꢀfield shift of imineꢀNH
Fig.10. Comparable pictogram of blue shift transition in electronic
spectra in form of energy difference.
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5RA08024J
of DFC-EN-p-Ph-NO2. When DFC-EN-p-Ph-NO2 (10 ꢁM) was
treated with HCT116 cells for 30 min at 37°C, it did not show
intracellular fluorescence (Fig. 13). The gradual increase in the blue
(for Al3+) and green (for PPi) fluorescence was observed with
increase in concentration of Al3+ and PPi, respectively. When DFC-
EN-p-Ph-NO2 (10 ꢁM ) form 1:1 intra cellular complex with Al3+ (10
ꢁM, 30 min at 37°C) it gives intense blue fluorescence. However,
when DFC-EN-p-Ph-NO2 (10 ꢁM) was treated with PPi (10 ꢁM, 30
min at 37°C) green fluorescence was observed intracellularly, but
no blue fluorescence suggesting that DFC-EN-p-Ph-NO2 +PPi
complex shifts emission wavelengths to green region (red shift). In
another experiment DFC-EN-p-Ph-NO2 –Al3+ ensemble (10 ꢁM
each) when treated with 10 ꢁM PPi the blue fluorescence of DFC-
EN-p-Ph-NO2 –Al3+ complex is almost vanishes, but in presence of
large excess of PPi green fluorescence corresponding to DFC-EN-p-
Ph-NO2 –PPi ensemble becomes visible. This suggests that DFC-
EN-p-Ph-NO2 could form optimum stable fluorescence complex
when incubated with either PPi or Al3+ ions separately. The dual
fluorescence sensory behaviour of DFC-EN-p-Ph-NO2 towards Al3+
and PPi was dependent on the ion to which it binds thereby opening
up its vast application towards biological sensing of these ions with
good photo stability and very low cytotoxicity at low concentration
region (10 ꢁM). Thus DFC-EN-p-Ph-NO2 facilitates the selective
sensing of either PPi or Al3+ and serves as the biomonitoring and
cell imaging tool for PPi and Al3+.
Fig.11. Frontier molecular orbitals involved in the UVꢀVis absorption
of DFC-EN-p-Ph-NO2.
Finally, to understand the selective interaction of DFC-EN-p-Ph-NO2
with PPi over ATP and Pi DFT calculations were performed to attain
the optimized geometry of DFC-EN-p-Ph-NO2 and its complexes
with PPi, ATP and Pi (2) (Fig. S11). From this study, it is clear that
the energy gap between the highest occupied molecular orbital
(HOMO) and the lowest unoccupied molecular orbital (LUMO) are
3.08. 3.48. 2.84 and 1.66 eV for free ligand and its complexes with
Pi, ATP, and PPi, respectively. It is to be noted that DFC-EN-p-Ph-
NO2–PPi has the lowest energy gap giving rise to a more stable
species,30 while DFC-EN-p-Ph-NO2–ATP has next lower energy gap
and this is reflected by substantial change in FI in case of former
and very low FI change in case of later. In case of DFC-EN-p-Ph-
NO2–Pi no observable change in FI was observed.
.
Fig.12. Frontier molecular orbitals involved in the UVꢀVis absorption
of DFC-EN-p-Ph-NO2+Al3+
Conclusion
Cell imaging experiments
In summary, we have successfully developed a new dual channel
chemical sensor for the detection of Al3+ and pyrophosphate ions
(PPi) together or separately. The probe DFC-EN-p-Ph-NO2 or DFC-
EN-p-Ph-NO2–Al3+ ensemble showed a highly sensitive and
The obvious excellent sensing capability of DFC-EN-p-Ph-NO2
towards PPi and Al3+ were further scrutinized in HCT116 cell lines.
The cytotoxic effect of DFC-EN-p-Ph-NO2 towards HCT116 cells
was determined by MTT assay which showed no severe cytotoxicity
till 60 ꢁM (<30% cytotoxicity) of DFC-EN-p-Ph-NO2 and more than
89.11% cell viability was observed at 10 ꢁM (Viability curve (Fig.
S12)). Hence further experiments were carried out with safer 10 ꢁM
2ꢀ
selective response to PPi in presence of other anions like HPO4 ,
2ꢀ
ꢀ
ꢀ
ꢀ
PO43−, ATP, AcO−, Cl−, Br−, F−, SO4 , SCNꢀ and N3 , AsO4 , S2O4
ꢀ
and NO2 at pH 7.4 by fluorescent change in organoꢀaqueous
solutions. In particular, the fluorescent detection of PPi was not
interfered by the presence of 400 ꢁM of ATP or Pi. Overall results
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indicate that the dual signal chemosensing of PPi by DFC-EN-p-Ph- imaging studies of these ions. The obvious excellent sensing
NO2 or DFC-EN-p-Ph-NO2 –Al3+ ensemble may contribute to the capability of DFC-EN-p-Ph-NO2 towards PPi and Al3+ were further
development of more efficient and useful methods for detecting PPi scrutinized in HCT116 cell lines. The modes of binding between
in biological systems. Its solubility in bioꢀcompatible solvent DFC-EN-p-Ph-NO2 with Al3+ and PPi were established by DFT
(DMSO/H2O 1:99 v/v) and cell permeability with no or negligible calculations.
cytotoxicity provide good opportunity towards inꢀvitro/inꢀvivo cell
Fig. 13: The phase contrast and fluorescence images of HCT116 cells captured (40X) after cells were preincubated with DFC-EN-p-Ph-
NO2 for 30min at 37ºC in the incubator followed by washing with 1XPBS for 3 time and allowed to treated with 0ꢁM, 4ꢁM, 6ꢁM, 8ꢁM and
10ꢁM Al3+ (ESꢀI), PPi (ESꢀII) and Al3++PPi (ESꢀIII) for 30 min at 37°C. The image shows the strong blue florescence when DFC-EN-p-Ph-
NO2 complexes with Al3+ and green fluorescence with PPi in blue and green filter respectively. The gradual increase in the blue (for Al3+) and
green (for PPi) fluorescence was observed with increase in concentration of Al3+ and PPi. ES; Experimental Set, PC; Phase Contrast, BF;
Blue filter, GF; Green filter. Cell imaging studies were carried out three times.
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Acknowledgement. Financial supports from CSIR (Ref. 13 S. Watchasit, A. Kaowliew, C. Suksai, T. Tuntulani, W.
02(2490)/11/EMRꢀII) and DST (Ref. SR/S1/ICꢀ20/2012) New Delhi
are gratefully acknowledged.
Ngeontae and C. Pakawatchai, Tetrahedron Lett.,2010, 51,
3398.
14 S. Mizukami, T. Nagano, Y. Urano, A. Odani and K. Kikuchi, J.
Am. Chem. Soc., 2002, 124, 3920.
15 K. M. K. Swamy, S. K. Kwon, H. N. Lee, S. M. S. Kumar, J. S.
Kim and J. Yoon, Tetrahedron Lett., 2007, 48, 8683.
16 N. Shao, J. Jin, G. Wang, Y. Zhang, R. Yang and J. Yuan,
Chem. Commun., 2008, 1127.
Supporting Information Available. Electronic Supplementary
Information (ESI) available: experimental details regarding the
synthesis and characterization of the ligand including
spectroscopic
details.
For
ESI
format
see
DOI: 10.1039/c000000x/
17 C. M. G. dos Santos and T. Gunnlaugsson, Dalton Trans.,
2009, 24, 4712.
18 C. R. Loran, J.ꢀM. Kim, S. ꢀY. Chung, J. Yoon and K.ꢀH. Lee,
Analyst, 2010, 135, 2079.
19 S. K. Kim, D. H. Lee, J.ꢀI. Hong and J. Yoon, Acc. Chem. Res.,
2009, 42, 23.
Authors’ Address
a
Department of Chemistry, Jadavpur University, Kolkata 700 032,
India, Fax: 91ꢀ33ꢀ2414ꢀ6223, Eꢀmail: m_ali2062@yahoo.com
Molecular & Human Genetics Division, CSIRꢀIndian Institute of
Chemical Biology, 4 Raja S.C. Mallick Road, Kolkataꢀ700032, India
b
20 S. Mizukami, T. Nagano, Y. Urano, A. Odani and K. Kikuchi, J.
Am. Chem. Soc., 2002, 124, 3920.
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