A single probe to sense Al(iii) colorimetrically and Cd(ii) by turn-on fluorescence in physiological conditions and live cells, corroborated by X-ray crystallographic and theoretical studies

Article abstract of DOI:10.1039/c4dt01433b

A pyridine-2-carbohydrazide functionalized conjugated fluorophoric Schiff base ligand (L₁) specifically senses Al³⁺ and Cd²⁺ ions through significant changes in its absorption and emission spectral behavior, respectively, in physiological conditions. The spectral changes are in the visible region of the spectrum and thus facilitate naked eye detection. Apart from the visible changes, an in-field device application was demonstrated by sensing these ions in paper strips coated with L₁. The crystal structure of the L₁-Cd complex provided additional insight of the metal coordination attributes of L₁. Interestingly, fluorescence microscopic studies demonstrated that the ligand L₁ could also be used as an effective probe in imaging experiments for the detection of intracellular Cd²⁺ ions in HeLa cells, without any toxicity to these model human cells. This journal is

Full text of DOI:10.1039/c4dt01433b

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Journal Name
RSCPublishing
ARTICLE
A Single Probe to Sense Al(III) Colorimetrically and
Cd(II) by TurnꢀOn Fluorescence in Physiological
Conditions and Live Cells: Corroborated by Xꢀray
crystallographic and Theoretical studies
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Chirantan Kara, Soham Samantaa, Sudeep Goswamib, Aiyagari Ramesh*b and
Gopal Das*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
A pyridineꢀ2ꢀcarbohydrazide functionalized conjugated fluorophoric Schiff base ligand L1
specifically sense Al3+ and Cd2+ ions through significant changes in their absorption and
emission spectral behavior, respectively, in physiological condition. The spectral changes are
in the visible region of the spectrum and thus facilitate naked eye detection. Apart from the
visible changes, an inꢀfield device application was demonstrated by sensing these ions in paper
strips coated with L1. The crystal structure of the L1ꢀCd complex provided additional insight
of the metal coordination attribute of L1. Interestingly, fluorescence microscopic studies
demonstrated that the ligand L1 could also be used as an effective probe in imaging
experiments for detection of intracellular Cd2+ ions in HeLa cells without any toxicity to these
model human cells.
developing Cd2+ꢀselective sensors that can distinguish Cd2+
from Zn2+ with high sensitivity and selectivity under
physiological conditions.
Introduction
Chemosensors for selective detection of various biologically
and environmentally important metal ions have recently
attracted great attention. Among various heavy metal ions
cadmium ions are banned in electrical and electronic equipment
by the European Union’s Restriction on Hazardous Substances
(RoHS) directive due to their adverse effect to human health.1
Cadmium metal ion is also not biodegradable, thus can be
easily accumulated in the environment, resulting in
contaminated food and water.2 Cadmium is also an extremely
toxic and carcinogenic metal.3 Although intake of cadmium
may also take place through smoking and food, but inhalation
of cadmiumꢀcontaining dust is the major route. Cadmium is
generally found in electric batteries, pigments in plastics,
electroplated steel, and so on.4 A high exposure level of
cadmium is related to damage of liver and kidneys, increased
risks of cardiovascular diseases, and cancer mortality.
Therefore, cadmium was listed as number 7 on ATSDR’s
“CERCLA Priority List of Hazardous Substances”5 and may
cause acute and chronic toxicity.
In fact, there are some fluorescent sensors that have been
recently reported for detection and analysis of cadmium metal
ions.6 However, there are only a few fluorescent sensors that
can detect cadmium with a fluorescence signal at higher
wavelength (orange or red) region of the spectra,6 and even rare
examples are available in living cells.7 So far, the greatest
challenge for detecting Cd2+ comes from the interference of
other transition metal ions like Zn2+, as they are in the same
group of the periodic table and hence exhibit similar chemical
properties. This sensing impediment underscores the need for
Along with cadmium, aluminium is also one of the most
biologically and environmentally relevant metal. Aluminium is
the third most abundant element (after oxygen and silicon), and
the most abundant metal, in the Earth's crust. Aluminium is
generally found in most animal and plant tissues and in natural
waters in its ionic form Al3+. Frequent use of aluminium in
water treatment, food additive, pharmaceutical products,
occupational dusts and cooking utensils results in moderate
increase in the Al3+ concentration in food. Tolerable weekly
aluminium dietary intake in the human body is estimated to be
7 mg/kg body weight.8 After absorption Al3+ is carried by the
plasma proteins and distributed in all tissues of human and
animals and eventually accumulate in bone, causing softening
of the bone, atrophy, and even aberrance.9 The presence of Al3+
ions have been implicated in the damage of the central nervous
system and is also thought to be involved in neurodegenerative
ailments such as Alzheimer’s disease and Parkinson’s
syndrome.9 Considering the physiological relevance and
biomedical implications of Al3+, there is significant interest in
developing selective and sensitive Al3+ sensors. Until recently,
only a few fluorescent chemosensors have been developed for
detection of Al3+.10ꢀ13
In recent years, fluorescent chemosensors have attracted
significant interest because of their potential use in medicinal
and environmental research. The most commonly employed
method used for metal ion detection is the designing of ligand
molecule containing an electron donating group at one end and
a metal coordination site at the other end, binding of metal ions
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to the ligand initiate the flow of electron from the electron residue was washed thoroughly with methanol to isolate L1 in
donating site through conjugated pi electron system, which is pure form with 68% yield (the yield was calculated based on
1
accompanied by an observable change in the electronic or the starting reagents). H NMR [600 MHz, CDCl3, SiMe4, J
fluorescence spectra of the ligand.
(Hz), δ (ppm)]: 10.81 (1H, s), 8.55 (1H, d, j=4.2), 8.29 (1H, d,
Most fluorescent sensors for Al3+ as well as Cd2+ possess good J=8.4), 7.98 (1H, d, J=9), 7.86ꢀ7.89 (1H, m), 7.45ꢀ7.47 (1H, m),
selectivity, but demonstration of these sensor for inꢀfield device 7.36 (2H, d, J=9), 6.96 (1H, q, J=9, J=15.6), 6.85 (1H, d,
application are scarcely reported. In our continuous endeavour J=15.6), 6.67 (2H, d, J=8.4), 2.99 (6H, s,). 13C NMR [150 MHz,
to develop sensor for various analytes,
14
herein, we CDCl3, SiMe4, δ (ppm)]: 159.99, 151.51, 151.21, 149.41,
demonstrate the cadmium and aluminium sensing capabilities 148.17, 141.22, 137.71, 128.78, 126.72, 124.08, 123.02,
of
a pyridineꢀ2ꢀcarbohydrazide functionalized conjugated 120.54, 112.24, 40.36. ESIꢀMS (positive mode, m/z). Calcd for
fluorophoric Schiff base ligand L1 (Scheme 1) and the C17H18N4O: 294.148. Found: 295.155 (M + H+). Anal. Calcd.
absorption and fluorescence behaviour of L1 upon metal for C17H18N4O (294.15): C 69.13, H 6.48, N 18.97, O 5.42;
complexation. The strong selectivity and inꢀfield application found C 69.10, H 6.45, N 19.08, O 5.37.
possibility of the developed sensor is verified by using it in
Synthesis of L2
paper strips. We have also demonstrated that the developed
sensor as well as the ligandꢀcadmium complex was nonꢀtoxic
and the ligand L1 could readily detect the presence of
intracellular Cd2+ ions in live HeLa cells via a characteristic
fluorescence switch ON mechanism.
Benzohydrazide was prepared following a literature method.16
Benzohydrazide
(Dimethylamino)cinnamaldehyde (175 mg, 5 mmol) were
dissolved in mL of methanol. To this was added
(136
mg,
5
mmol)
and
4ꢀ
5
approximately 2 drops of acetic acid, and the resulting solution
was stirred at room temperature for 10 h. A yellow precipitate
was obtained, which was collected through filtration. The
residue was washed thoroughly with methanol to isolate L2 in
pure form with 82% yield (the yield was calculated based on
Experimental section
General information and materials
All the materials for synthesis were purchased from commercial
suppliers and used without further purification. The absorption
spectra were recorded on a PerkinꢀElmer Lamdaꢀ750 UVꢀ
visible spectrophotometer using 10 mm path length quartz
cuvettes in the range of 300–800 nm wavelengths, while the
1
the starting reagents). H NMR [400 MHz, DMSOꢀd6, J (Hz), δ
(ppm)]: 11.61 (1H, s), 8.18 (1H, d, j=8.8), 7.89 (2H, d, j=7.6),
7.57 (1H, d, j=6.8), 7.51 (2H, t, j=6.8), 7.45 (2H, d, j=8.4), 6.91
(1H, d, j=16), 6.78 (1H, q, j=9.2, j=15.6), 6.70 (2H, d, j=8),
2.94 (6H, s). 13C NMR [100 MHz, DMSOꢀd6, δ (ppm)]: 162.75,
150.74, 139.87, 133.67, 131.56, 128.44, 127.57, 123.68,
120.56, 112.03, 40.20. ESIꢀMS (positive mode, m/z). Calcd for
C18H19N3O: 293.153. Found: 294.161 (M + H+). Anal. Calcd.
for C18H19N3O (293.153): C 73.44, H 6.85, N 14.27, O 5.44;
found C 73.40, H 6.83, N 14.40, O 5.37.
fluorescence measurements were carried on
a
Horiba
Fluoromaxꢀ4 spectrofluorometer using 10 mm path length
quartz cuvettes with a slit width of 5 nm at 298 K. NMR spectra
were recorded using Bruker 600 MHz instrument and Varian
FTꢀ400 MHz instrument. The mass spectra of L1, L1ꢀCd and
L1ꢀAl complex was obtained using Waters QꢀToF Premier mass
spectrometer. The chemical shifts were recorded in parts per
million (ppm) on the scale. The following abbreviations are
Synthesis of L3
1
L3 was synthesized following a method previously reported in
used to describe spin multiplicities in H NMR spectra: s =
literature.17
singlet; d = doublet; t = triplet; m= multiplate. Elemental
analyses were performed with a Perkin Elmer 2400 elemental
analyzer.
Synthesis of Cadmium complex for crystallization
A solution of L1 in DMF was stirred and refluxed in presence
of 1 equivalent of Cadmium chloride. The color of the solution
changes to dark yellow after addition of the cadmium salt. The
stirring is continued in reflux condition for 2 hours until the
solution color changed to dark reddish. The resulting solution
1
was cooled, filtered and kept for crystallization. H NMR [400
MHz, DMSOꢀd6, J (Hz), δ (ppm)]: 12.08 (1H, s), 8.69 (1H, s),
8.37 (1H, d, j=8.8), 8.10 (1H, d, j=7.6), 8.04 (1H, t, j=7.2), 7.65
(1H, s), 7.43 (2H, d, j=8.4), 6.92 (1H, d, j=16), 6.81 (1H, d,
j=8.8), 6.70 (2H, d, j=7.6) 2.50 (6H, s). ESIꢀMS (positive
mode, m/z). Calcd for (C17H19N4O)2CdCl2: 774.152. Found:
737.163 [(C17H19N4O)2CdCl]+.
Scheme 1 Synthetic scheme for the ligand L1, L2 and L3
Synthesis of L1
UV−Vis and fluorescence spectral studies
Stock solutions of various ions (1 × 10−3 mol·L−1) were
prepared in deionized water. A stock solution of L1 (1 × 10−3
mol·L−1) was prepared in DMSO. The solution of L1 was then
diluted to 1 × 10−5 mol·L−1 with CH3OH/aqueous HEPES
buffer (5 mM, pH 7.3; 1:4 v/v). In each titration experiments a
1 × 10−3 L solution of L1 (1 × 10−5 mol·L−1) was taken in a
quartz optical cell of 1 cm optical path length, and then the ion
Picolinohydrazide was prepared following
a
literature
method.15 Picolinohydrazide (137 mg, 5 mmol) and 4ꢀ
(Dimethylamino)cinnamaldehyde (175 mg, 5 mmol) were
dissolved in
5 mL of methanol. To this was added
approximately 2 drops of acetic acid, and the resulting solution
was stirred at room temperature for 10 h. A yellow precipitate
was obtained, which was collected through filtration. The
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stock solutions were added into the optical cell gradually by intensity data were collected using a Bruker SMART APEXII
using a micropipette. Spectral data were recorded at 1 min after CCD diffractometer, equipped with a fineꢀfocus 1.75 kW
the addition of the ions. In selectivity experiments, the test sealedꢀtube Mo Kα radiation (λ = 0.710 73 Å) at 298(3) K, with
samples were prepared by placing appropriate amounts of the increasing ω (width of 0.3° frameꢀ1) at a scan speed of 5 s
cations stock into 2 mL of solution of L1 (2 × 10−5 mol·L−1). frameꢀ1. SMART software was used for data acquisition. Data
For fluorescence measurements, excitation was provided at 405 integration and reduction were undertaken with SAINT and
nm, and emission was collected from 420 to 750 nm.
XPREP19 software. Multiscan empirical absorption corrections
were applied to the data using the program SADABS.20
Structures were solved by direct methods using SHELXSꢀ9721
and refined with fullꢀmatrix least squares on F2 using
SHELXLꢀ97.22 All nonꢀhydrogen atoms were refined
anisotropically. The hydrogen atoms were located from the
difference Fourier maps and refined. Structural illustrations
have been drawn with ORTEPꢀ3 for Windows.23
Evaluation of the binding constant for the formation of L1·Cd2+
and L1·Al3+
Receptor L1 with an effective concentration of 10.0 × 10ꢀ6 M in
a Methanol/aqueous HEPES buffer (5 mM; 1:4, v/ v; pH 7.3)
was used for the spectroscopic titration studies with a Cd2 or
+
Al3+ solution. A stock solution of Cd(NO3)2 or Al(NO3)3,
having a concentration of 0.2 × 10ꢀ3 M in an Methanol/aqueous
HEPES buffer (1:4, v/v; pH 7.3) solution was used. The
Table 1. Crystallographic Parameters and Refinement Details of L1 and L1ꢀ
Cd Complex
+
effective Cd2 or Al3+ concentration was varied between 0 and
5 × 10ꢀ5 M for this titration. The solution pH was adjusted to
7.3 using an aqueous HEPES buffer solution having an
effective concentration of 5 mM.
Parameters
Formula
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
β (deg)
L1
L1ꢀCd
C34H40CdCl2N8O4
808.05
Monoclinic
C2/c
18.0309(4)
10.7197(4)
20.3059(6)
111.574(2)
3649.88(19)
4
C17H18N4O
294.35
Monoclinic
P21
5.9895(4)
14.7978(8)
17.8142(14)
96.481(6)
1568.81(18)
4
Binding constants using spectroscopic titration data
The binding constant for the formation of the respective
complexes were evaluated using the Benesi
−
Hildebrand (B−H)
plot (eq 1 and eq 2).18
1/(AꢀA0)=1/{K(AmaxꢀA0)C}+1/(AmaxꢀA0)
1/(IꢀI0)=1/{K(ImaxꢀI0)C}+1/(ImaxꢀI0)
(1)
(2)
V (Å)
Z
A0 is the absorbance of L1 at absorbance maximum (λ = 510
nm), A is the observed absorbance at that particular wavelength
in the presence of a certain concentration of the metal ion (C),
Amax is the maximum absorbance value that was obtained at λ =
510 nm during titration with varying metal ion concentration, K
is the binding constant (Mꢀ1) and was determined from the slope
of the linear plot, and C is the concentration of the Al3+ ion
added during titration studies.
I0 is the emission intensity of L1 at emission maximum (λ = 578
nm), I is the observed emission intensity at that particular
wavelength in the presence of a certain concentration of the
metal ion (C), Imax is the maximum emission intensity value that
was obtained at λ = 578 nm during titration with varying metal
ion concentration, K is the binding constant (Mꢀ1) and was
determined from the slope of the linear plot, and C is the
concentration of the Cd2+ ion added during titration studies.
T (K)
ꢁ (cmꢀ1)
298(2)
0.081
298(2)
0.793
dcal (g cmꢀ3)
crystal dimens (mm3)
no. of reflns collected
no. of unique reflns
no. of params
R1; wR2 (I>2σ(I))
R1; wR2(all)
GOF (F2)
1.246
0.25×0.16×0.14
2513
5103
401
0.0776; 0.2489
0.1564; 0.3180
0.932
1.470
0.26×0.20×0.16
3086
4158
236
0.0308;0.0958
0.0422; 0.1066
0.783
CCDC No.
989403
989402
Computational Methods
Full geometry optimizations were carried out using the density
functional theory (DFT) method at the Beckeꢀ3ꢀLeeꢀYangꢀParr
(B3LYP)24 level for the ligand L1 and its octahedral Al3+
complexes ( for both the possibilities; ‘N’ coordinated and ‘O’
coordinated). The 6ꢀ31+G (d,p) basis set was assigned for all
the elements. All calculations were performed with Gaussian03
program with the aid of the GaussView visualization program.
Detection limit for Cd2+ and Al3+
The detection limit was calculated based on the UVꢀVis
titration for Cd2+ and fluorescence emission in case of Al3+. The
fluorescence emission spectrum of L1 was measured ten times
and the standard deviation of blank measurement was achieved.
To gain the slop, the UVꢀVis absorbance (or fluorescence
emission in case of Cd2+) at 510 nm (or at 578 nm in case of
Cd2+) and was plotted as a concentration of the corresponding
metal ion concentration. The detection limit was calculated
with the following equation:
Cytotoxic effect on HeLa cells
The cytotoxic potential of compound L1 and L1ꢀCd complex
was ascertained by an MTT assay following the manufacturer
instruction (SigmaꢀAldrich, MO, USA). HeLa cells were
initially propagated in a 25 cm2 tissue culture flask in
Dulbecco's Modified Eagle Medium (DMEM) supplemented
with 10% (v/v) fetal bovine serum (FBS), penicillin (100
ꢁg/mL) and streptomycin (100 ꢁg/mL) in a CO2 incubator.
Prior to MTT assay, cells were seeded into 96ꢀwell plates
(approximately 104 cells per well) and various concentrations of
compound L1 and L1ꢀCd complex (15, 30, 45 and 60 ꢁM) made
Detection limit = 3σ/k
Where σ is the standard deviation of blank measurement, k is
the slop between the ratio of UVꢀVis absorbance (or
fluorescence emission) versus metal ion concentration.
Xꢀray Crystallography
In each case, a crystal of suitable size was selected from the
mother liquor and immersed in silicone oil, and it was mounted
on the tip of a glass fibre and cemented using epoxy resin. The
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in DMEM were added to the cells and incubated for 24 h. accompanied by 40 nm red shift of the absorption maxima but a
Solvent control samples (cells treated with DMSO alone) and significant change in UVꢀvis spectral pattern was observed only
cells treated with Cd(NO3)2 alone were also included in parallel in presence of Al3+, among all the other metal ions used (Figure
sets. Following incubation, the growth media was removed and 1A). Upon addition of Al3+, the intensity of the absorption
fresh DMEM containing MTT solution was added. The plate bands of L1 at 390 nm decreased with a concomitant
was incubated for 3ꢀ4 h at 37oC. Subsequently, the supernatant appearance of two new bands at 330 nm and at 510 nm. The
was removed and the insoluble colored formazan product was change in the UVꢀvis spectra is also well supported by a visual
solubilized in DMSO and its absorbance was measured in a change of the receptor solution from yellow to dark red on
microtitre plate reader (Infinite M200, TECAN, Switzerland) at addition of nearly 1.0 equivalent of Al3+ ions (Figure 1B inset).
550 nm. The assay was performed in six sets for each We have also performed the UVꢀvis titration experiment with
concentration of compound L1 and L1ꢀCd complex. Data incremental amount of Al3+ ions, wherein two wellꢀdefined
analysis and calculation of standard deviation was performed isosbestic points at 348 and 445 nm were observed (Figure 1B),
with Microsoft Excel 2010 (Microsoft Corporation, USA). For which clearly suggested a prominent conversion of L1 into the
statistical analysis, a one way analysis of variance (ANOVA) L1ꢀAl complex. The complex formed between L1 and Al3+ was
was performed using Sigma plot.
observed to be 1:1 in stoichiometry, which was established with
the help of job’s plot (supporting information figure S6) based
on the changes in absorbance at 510 nm. This stoichiometry in
the solution state was also supported by ESIꢀMS studies
(supporting information figure S7), which indicated the
presence of a molecularꢀion peak at m/z 445.05, corresponds to
the mass of [L1+Al +3Cl+H2O+H]+. The binding constant for
the formation of L1ꢀAl complex was also calculated on the
basis of change in absorbance at 510 nm by considering a 1:1
binding stoichiometry (supporting information figure S8). The
Cell culture and imaging studies
HeLa cells were initially propagated in a 25 cm2 tissue culture
flask as described earlier and then seeded into a 6 well plate and
incubated at 370 C in a CO2 incubator for 3 days. Subsequently
the cells were washed three times with sterile phosphate
buffered saline (pH 7.4) and incubated with 10 ꢁM L1 in
DMEM at 37 °C for 1 h in a CO2 incubator. The image of the
cells was then recorded using an epifluorescence microscope
(Nikon Eclipse Ti). The cells were again washed thrice with
sterile PBS (pH 7.4) to remove excess L1, and then incubated in
sterile PBS (pH 7.4) with 20 ꢁM Cd(NO3)2 for 1 h and the
image of the cells were again recorded using epifluorescence
microscope.
binding constant (K) determined by the BꢀH method was found
to be 5 × 104 Mꢀ1. It is significant to mention that the detection
limit of L1 for Al3+ ions was found to be 8.7 µM (supporting
information figure S9) which is lower than tolerable weekly
aluminium dietary intake in an average human body.8 The
selective change in the absorbance spectra of L1 can be
attributed to the high positive charge of the metal ion. On
complexion with L1, Al3+ significantly attracts the electron
cloud from the N,N dimethyl moiety via conjugated πꢀelectron
system and the probability of Internal charge transfer (ICT) also
increases resulting in formation of a red shifted new band in the
spectra.
Results and Discussions
UVꢀvis spectroscopic studies of L1 in presence of metal ions
For a theoretical appraisal on the selective chromogenic
response of L1 toward Al3+, the Density Functional Theory
Calculation (DFT) of L1 and its Aluminium complex were
carried out. The electronic spectrum of L1 alone showing
absorbance maximum at 390 nm is mainly generated from the
transition of HOMO to LUMO. However as both the Job’s plot
and mass spectrum indicated formation of a 1:1 L1ꢀAl complex;
we have performed the structure optimization of L1ꢀAl complex
to explore the most suitable binding mode between L1 and Al3+.
As for Al3+ two probable coordination sites are available, the
first one involving the pyridine nitrogen (Structure
i in figure
2A) and the second one involving the carbonyl oxygen
(structure ii in figure 2A), theoretical calculations were done for
both the possibility. Structure ‘ii’ was found to be 8.01 Kcal
more stable than structure ‘i’ (supporting information table S1).
Thus it can be argued here that chelation of Al3+ with L1
involving the carbonyl oxygen and the imine nitrogen led to the
formation of a highly stable complex. The DFT calculation
revealed that there is a reasonable decrease in the HOMO to
LUMO energy gap from L1 to its Aluminium complex (figure
2B) which is in agreement with the subsequent generation of
Fig. 1 (A) UVꢀVis absorption spectra of receptor L1 (10 ꢁM) observed upon
addition of 5 equivalent metal ions (perchlorate or nitrate salts of Na+, Mg2+, Cr3+
,
Hg2+ Cu2+ Pb2+ Zn2+ Fe3+ Co2+, Ni2+ Cd2+ Yb3+ Tb3+ and Al3+) in
, , , , , , , , a
CH3OH/aqueous HEPES buffer (5 mM, pH 7.3; 1:4 v/v). (B) UVꢀVis titration
spectra of L1 (10 ꢁM) upon incremental addition of Al(NO3)3 in CH3OH/aqueous
HEPES buffer (5 mM, pH 7.3; 1:4 v/v). Inset: Changes in the absorbance at 510
nm with incremental addition of Al3+ and visual change in the solution colour
after addition of metal ions.
UV−vis spectra recorded for L1 in CH3OH/aqueous HEPES
the red shifted new absorbance peak on addition of Al3+ to L1
.
buffer (5 mM, pH 7.3; 1:4, v/v) indicated an absorption
maximum at 390 nm, which may have originated due to the
intraꢀmolecular πꢀπ* charge transfer (CT) transition. The
selectivity of L1 was verified with perchlorate or nitrate salts of
Na+, Mg2+, Cr3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe3+, Co2+, Ni2+, Cd2+,
Yb3+, Tb3+ and Al3+ in CH3OH/aqueous HEPES buffer (5 mM,
pH 7.3; 1:4 v/v). Although other metal ions like Cd, Ni, Zn, Pb
and Co exhibit some binding with the receptor L1 which is
The comparison between the structure of L1 and L1ꢀAl complex
clearly revealed that on chelation with the Al3+, ligand L1
gained perfect planarity with an extended conjugation
(supporting information figure S10). Thus, undoubtedly the
formation of the highly stable L1ꢀAl complex followed by
extension in the conjugation and increases in the probability of
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charge transfer initiated this unusual colorimetric response. 3A, a solution of L1 (10.0 × 10−6 M) exhibited a low intensity
Selected orbitals and their corresponding energies for both L1 emission maxima at 554 nm when excited at 405 nm. Addition
and L1ꢀAl complex, which are likely to be critical in the optical of Cd2+ to this receptor solution induced a dramatic increase in
spectral outcome are provided in supporting information the fluorescence intensity along with a significant red shift of
(supporting information table S2 and table S3).
the emission maxima to 578 nm. The increase in fluorescence
was also observed under an UV lamp wherein there was a sharp
change in the solution fluorescence from colorless to orange in
presence of Cd2+ ions (Figure 3A inset)
. It can also be observed
from Figure 3A that the metalꢀligand binding induced switchꢀ
ON fluorescence of L1 was entirely selective towards Cd2+ ions,
as no significant fluorescence change of L1 occurred even in the
presence of excess of other metal ions. To further understand
the properties of L1 as a receptor for Cd2+, a titration of the
receptor was performed with increasing concentration of Cd2+.
As evident in Figure 3B the fluorescence intensity of a 10 ꢁM
solution of L1 was enhanced systematically with incremental
addition of Cd2+ ions, which also confirmed that receptor L1
exhibited a high sensitivity toward Cd2+. The 1:1 stoichiometry
of the L1ꢀCd was established from the measurements of
emission intensity as a function of Cd2+ concentration (inset in
Figure 3B), where a clear bend of the curve was observed at 1.0
equivalent of added Cd2+.
This stoichiometry in the solution state was also confirmed with
the help of job’s plot (supporting information figure S6) and
further supported by ESIꢀMS studies (supporting information
figure S13), which indicated the presence of a molecularꢀion
peak at m/z 443.02, corresponds to the mass of [L1 + Cd2+
+
Cl]. The binding constant for the formation of L1ꢀCd complex
was calculated on the basis of change in emission at 578 nm by
considering
a
1:1 binding stoichiometry (supporting
information figure S7). The binding constant (K) determined by
the BꢀH method was found to be 1.6 × 105 Mꢀ1. The detection
limit of L1 for Cd2+ ions was found to be 1.2 µM (supporting
information figure S9).
Fig. 2 (A) Optimized structures of
binding modes. (B) Energy diagrams of HOMO and LUMO orbitals of
1–Al3+ complex (structure
) calculated at the DFT level using a B3LYP/6ꢀ
31+G(d,p) basis set.
L
1–Al3+ complex with two different possible
1 and the
L
L
i
The change in emission spectral behavior of L1 in presence of
Cd2+ can be explained by chelationꢀenhanced fluorescence
(CHEF). Although L1 is a conjugated system but the metal
binding site and the N,Nꢀdimethyl aniline ring of L1 may not be
perfectly planar in the free ligand. Metal coordination renders
the system more planarity which in turn increases the rigidity of
the system25 and consecutively enhances the internal charge
transfer from the N,Nꢀdimethyl aniline moiety to the metal
binding cites. In absence of the metal ions there is no sufficient
pull for the electronic charge over the N, Nꢀ Dimethyl
substituted benzene ring and thus the fluorescence is quenched
due to lack of internal charge transfer throughout the system.
But binding of a suitable metal cation by the imine nitrogen and
the amide oxygen may drag the electronic charge from the N,
Nꢀ Dimethyl substituted benzene ring. Due to the metal ion
assisted electron pull and the N, Nꢀ Dimethyl inducted electron
push, the possibility of internal charge transfer throughout the
πꢀsystem increases; leading to a highly conjugated geometry
and a radical enhancement of the fluorescence intensity. To
further verify the significance of the conjugated chain length
and the pyridine unit in the sensing event of metal ions, we
Fluorescence spectroscopic studies of L1 in presence of metal
ions
Fig. 3 (A) Changes of the fluorescence emission of receptor L1 (10 ꢁM) observed
upon addition of 5 equivalent metal ions (perchlorate or nitrate salts of Na+, Mg2+
,
Cr3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe3+, Co2+, Ni2+, Cd2+, Yb3+, Tb3+ and Al3+) in a
CH3OH/aqueous HEPES buffer (5 mM, pH 7.3; 1:4 v/v). Inset: Visual change in
the solution fluorescence after addition of metal ions. (B) Fluorescence titration
have synthesized two control compounds L2 and L3
. L2 has a
similar structure to L1 but in place of a pyridine moiety in L1
a
spectra of L1 (10 ꢁM) upon incremental addition of Cd(NO3)2 (upto
equuivalent) in CH3OH/aqueous HEPES buffer (5 mM, pH 7.3; 1:4 v/v). Inset:
Changes in the emission intensity at 578 nm with incremental addition of Cd2+
2
benzene moiety is present. In case of L3 we had chosen N, N
dimethyl carboxaldehyde in place of the cinnamaldehyde
(Scheme 1). Fluorescence emission and UVꢀVis spectroscopic
studies with L2 and L3 revealed some interesting facts, which
help us to further understand the beauty of the ligand designing.
From fluorescence emission spectra of L2 we found that there
was no significant increase in the emission intensity with the
.
The selectivity of L1 for different metal ions (Na+, Mg2+, Cr3+,
Hg2+, Cu2+, Pb2+, Zn2+, Fe3+, Co2+, Ni2+, Cd2+, Yb3+, Tb3+ and
Al3+ in CH3OH/aqueous HEPES buffer) was also studied by
fluorescence emission spectroscopy. As evident from Figure
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tested metal ions, similarly UVꢀvis absorbance was also almost the pyridine moiety are significant prerequisites for the metal
unchanged upon addition of an excess amount of Cd2+/Al3+ to a sensing ability of L1. The pyridine ring basically controls the
solution containing L2 (supporting information figure S14 and selective optical response for the metal ions and the extended
figure S15). whereas in case of L3 there was a significant conjugation assist to shift the optical response to longer
increase in the emission intensity (at 520 nm) in presence of wavelength region of the spectrum. Quantum yield recorded for
Cd2+, although the response towards Zn2+ ions is much more L1 L3 and L1 in presence of 1 equivalent of Cd2+ ions are
, L2,
pronounced but perceivable change in presence of Cd2+ is 0.08, 0.02, 0.009 and 0.25 respectively. (Quinine sulfate in
sufficient enough to understand the significance of the pyridine 0.1N H2SO4 was used as the standard for quantum yield
moiety to control the optical response of the ligand towards measurements).
specific cations. Similar to the result obtained from the Further proof of the metal binding modes of the ligand was
fluorescence spectroscopy, UVꢀVis absorbance shows obtained from the Xꢀray crystallographic studies of L1ꢀCd
formation of a new peak (near 450 nm) in presence of Al3+ ions. complex.
Thus, from the above mentioned result it can be concluded that
the presence of both, the long conjugated geometry as well as
Fig. 4 (A) Thermal ellipsoid plot (50% probability level) of L1 (B) Packing diagram of
L1 (C) Thermal ellipsoid plot (50% probability level) of the L1ꢀCd complex. (D)
Packing diagram of L1ꢀCd complex. All hydrogen atoms were deleted for clarity
.
nitrogen, carbohydrazide oxygen, and chloride anion. Two
ligand molecules along with chloride ions form disordered
octahedral coordination geometry. The Cd2+–N bond
distance is 2.477 Å, the Cd2+ꢀClꢀ bond distance is 2.479 Å
and the distance between O and Cd2+ is of 2.365 Å. These
data demonstrate that the hydrazide moiety is mainly
involved in the capture of metal ions, while the
cinnamaldehyde moiety is responsible for the optical
response of the sensor. From Xꢀ ray crystallographic study
we found that 2:1 binding stoichiometry is favoured in solidꢀ
state, but in solution 1:1 stoichiometry is proved. The
discrepancy between the binding stoichiometry in the solidꢀ
state and solution is inꢀlieu with the known literature.26
However, repeated attempt to get Xꢀray quality crystal of
L1ꢀAl3+ complex was unsuccessful.
Xꢀray Crystallographic Analysis
Single crystals of L1 suitable for XRD analysis were
obtained from methanol, which crystallizes in monoclinic
space group P21 with Z = 4. The asymmetric unit contains
two symmetryꢀindependent L1 molecules (Z′ = 2), which
differ considerably in their torsions involving the conjugated
aliphatic ꢀCH=CHꢀCH=Nꢀ chain (Figure 4A). The solid
states structure of the metal complex L1ꢀCd was also
investigated by Xꢀray crystallography. Initially, we
attempted to crystallize various metal salts [Chloride and
nitrate salts of Cd2+ and Al3+] with L1 in a variety of
solvents. Indeed, the crystals suitable for crystallographic
analysis were obtained in DMF/methanol mixture. The
crystal structures and data of both the ligand and the
complex are shown in Figure 4, and Table 1. In the Cd
complex L1ꢀCd
,
each asymmetric unit contains one
MetalꢀIon Competition Studies
coordinating ligand, where Cd2+ is chelated by imine
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Fig. 5 (A) Normalized fluorescence responses of L1 (10 ꢁM) to various
cations in CH3OH/aqueous HEPES buffer (5 mM, pH 7.3; 1:4 v/v). The blue
bars represent the emission intensities of L1 in the presence of cations of
interest (50 ꢁM). The red bars represent the change of the emission that
occurs upon the subsequent addition of Cd2+ to the above solution; the
intensities were recorded at 578 nm. (B) Absorbance of L1 (10 ꢁM) at the
wavelength 510 nm in presencence of various cations in CH3OH/aqueous
HEPES buffer (5 mM, pH 7.3; 1:4 v/v). The blue bars represent the
absorbance of L1 in the presence of cations of interest (50 ꢁM/ 5 equivalent).
The red bars represent the change of the absorbance that occurs upon the
Fig. 6 Demonstration of the Al3+ and Cd2+ sensing in water by L1 coated
(25ꢁM solution) paper strips. Strength of the metal solutions used was 25ꢁM.
subsequent addition of Al3+ to the above solution
.
Intracellular sensing of Cd2+ by imaging studies
Given the significant physiological and toxicological
implications of cellular cadmium pool, there is considerable
motivation in developing probes for detecting intracellular
levels of cadmium. In our study, the results obtained in
solutionꢀbased fluorescence measurements with the ligand
L1 demonstrated the potential of the ligand to selectively
detect Cd2+ at low levels. However, to harness the potential
of the ligand L1 for sensing intracellular Cd2+ levels, it was
pertinent to initially determine the cytotoxic effect of L1 as
The individual emission response of L1 in presence of
different transitionꢀmetal ions had revealed a notable
selectivity of Cd2+ and Al3+ binding (Figure 1 & 2).
However, the most important criterion for a selective cation
probe is the ability to detect a specific cation in a complex
milieu of other competing ions. To validate this hypothesis,
the selectivity of L1 was further tested in presence of other
competing cations, which may interfere with the estimation
of Cd2+ or Al3+ (figure 5A). The emission intensity of Cd2+ꢀ
bound L1 was nearly unaltered in the presence of 5 equiv of
Na+, Mg2+, Cr3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe3+, Co2+, Ni2+ and
Al3+, indicating excellent selectivity for Cd2+ over these
competing cations. The response of L1 for Al3+ over other
competing metals ions was evaluated based on UVꢀVisible
absorbance at 510 nm. It was observed that sensitivity of L1
towards Al3+ in the presence of Cd2+ was relatively low but
presence of other metal ions did not affect the detection
(figure 5B).
well as the L1ꢀCd complex.
Application of L1 in Paper strips
The strong sensing potential of L1 for Al3+ and Cd2+ ions are
further verified by incorporation in paper strips. Sensing
paper strips were prepared by coating the L1 solution (in
chloroform) on a filter paper to demonstrate the inꢀfield
device application. As evident from figure 6 paper strips
coated with only L1 exhibited a moderate yellow emission
under a hand held UV lamp. However, after dipping it into
cadmium nitrate solution an intense orange fluorescence was
observed. The aluminium sensing behaviour of L1 under
visible light was also captured in a sharp change in colour of
the paper strip from yellow to red. Thus, the paper strips
dipped in a solution of L1 showed a turn on response similar
to the result obtained from the spectroscopic studies and was
selective to Cd2+ and Al3+. Although zinc is known to be
analogous to cadmium in spectroscopic behavior it was
interesting to observe that zinc failed to promote any turn
ON response neither with the paper strips, nor in the
fluorescence emission studies. To the best of our knowledge
this is the first report where a monomeric sensor molecule is
used to demonstrate the paper strip model that can
efficiently sense Cd2+ in presence of other metals including
Zn2+.
Fig. 7 MTT assay to determine the cytotoxic effect of compound L1 and L1ꢀ
Cd complex on HeLa cells.
A standard in vitro MTT assay suggested that at low
concentrations (upto 30µM) both L1 and L1ꢀCd complex
failed to exert any detrimental effect on the viability of
HeLa cells (Figure 7). The nonꢀtoxic nature of the ligand L1
holds significant implications in the context of sensing and
suggested that the developed sensor was not only selective
for Cd2+ but also amicable to nonꢀdestructive intracellular
sensing of the metal ion. Encouraged by the nonꢀtoxic
attribute of L1, our subsequent endeavor was to ascertain the
sensing potential of L1 for intracellular detection of Cd2+
through fluorescence microscopy.
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providing instrument facilities and DST−FIST for single
crystal Xꢀray diffraction facility. CK, SS, SG acknowledge
Department of Chemistry, Central instrumental facility and
IIT Guwahati for instrumental support and fellowship. We
thank Department of Biotechnology, Government of India
for a research grant (BT/01/NE/PS/08).
Notes and references
a
Department of Chemistry, Indian Institute of Technology Guwahati,
Assam, 781 039, India.
b
Department of Biotechnology, Indian Institute of Technology
Guwahati, Assam, 781 039, India.
1
Electronic Supplementary Information (ESI) available: H NMR, 13C
Fig. 8 Fluorescence microscopic images of HeLa cells after adding 10 ꢁM of
L
1 (panel aꢀc) and after subsequent treatment with 15 ꢁM Cd2+, (panel dꢀf).
NMR, mass spectra as characterization data of L1 and L1ꢀCd
complex. BenesiꢀHildebrand plot, Job’s plot, UVꢀvis spectra and
fluorescence spectra of L2 and L3, details of the result obtained from
DFT calculation.
When HeLa cells were treated with 10 µM L1, the cells
failed to exhibit any fluorescence (Figure 8B, Panel b).
Interestingly, when the cells were subsequently exposed to
15 µM Cd(NO3)2,
a
dramatic switchꢀON orange
1
The European Parliament and the Council of the European Union,
“Directive on the Restriction of the Use of Certain Hazardous
Substances in Electrical and Electronic Equipment”. 2002/95/EC.
W. De Vries, P. F. Romkens and G. Schutze, Rev. Environ.
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fluorescence manifested (Figure 8B, Panel e), indicating
selective sensing of Cd2+ in cells. The evenly distributed
intracellular fluorescence observed across HeLa cells
suggested that the ligand L1 could transit through the
membrane and pervade the cells and upon subsequent
addition of Cd(NO3)2, formation of the highly fluorescent
2
3
4
R. R. Lauwerys, A. M. Bernard, H. A. Reels and J.ꢀP. Buchet,
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L1ꢀCd complex resulted throughout the cell. It was also
observed that HeLa cells retained their typical
morphological trait during treatment with the ligand and
Cd(NO3)2 as evident from the bright field images of treated
cells (Figure 7B, Panels a and d). This observation also
suggested that the ligand L1 as well as L1ꢀCd complex were
not cytotoxic to HeLa cells, corroborating earlier results of
the MTT assay.
G. F. Nordberg, R. F. M. Herber and L. Alessio, Cadmium in the
Human Environment, Oxford University Press, Oxford, UK,
1992.
5
6
Agency for Toxic Substances and Disease Registry, 4770 Buford
Hwy
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Atlanta,
GA
30341
http://www.atsdr.cdc.gov/cercla/07list.html.
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Conclusion
In summary, we have developed a receptor L1 which
selectively sense Al3+ and Cd2+ ions with a switch ON
response respectively in optical and fluorescence spectra in
the visual region. Apart from the visible changes, an inꢀfield
device application was further demonstrated by sensing Al3+
and Cd2+ ions in paper strips coated with L1. Binding ability
of the receptor with Cd2+ was confirmed by single crystal Xꢀ
ray diffraction studies of the complex, which also supports
the strong interaction between the ligand and Cd2+ ions. The
fluorescence spectroscopic studies revealed that L1
displayed strong preference for Cd2+ ions over other
interfering ions including Zn2+. The observations of the
cytotoxicity assay and fluorescenceꢀbased cell imaging are
encouraging for future diagnostic applications of the sensor.
Given the fact that the ligand L1 and L1ꢀCd complex were
found to be nonꢀtoxic to cultured HeLa cells even after 24 h
interaction period, the cadmium probe L1 developed in the
present study holds interesting prospect in analytical and
biomedical domain applications.
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Graphical Abstract
Selective recognition of Al3+ and Cd2+ by UV-Vis and fluorescence based techniques using a
cinnamaldehyde functionalized conjugated ligand and their application in paper strip and live
cell imaging.