Rhodamine-based chromo-/fluorogenic dual signalling probe for selective recognition of HgII with potential applications for INHIBIT logic devices and cell-imaging studies

Article abstract of DOI:10.1002/ejic.201300809

A new rhodamine-based dual signaling probe (L³) has been found to display quick responses through visible colorimetric changes as well as fluorogenic properties on selective 1:1 binding to Hg²⁺, as delineated by absorption and fluorescence titrations as well as by Job′s method and ESI-MS⁺ studies. The fluorescent probe L³ displays a 252-fold fluorescence enhancement on binding to Hg²⁺. A Benesi-Hilderband fit of the absorption titration data gives K_d = (32.01 ± 0.74) μM and a 1:1 binding stoichiometry. Owing to its solubility in mixed organic/aqueous solvents as well as its cell permeability, L³ could be used for in vitro/in vivo cell imaging of Hg²⁺ with no or negligible cytotoxicity. The detection limit of Hg²⁺ was calculated by the 3σ method to be 9.28 ng L^-1. Moreover, the fluorescence of the L³-Hg²⁺ system was turned off and its red colour disappeared when KI was added. Thus, the OFF-ON-OFF fluorescence sensing behaviour of L³ observed in the presence of Hg²⁺ and I^- may find applications in devices with logic gate functions. A new rhodamine-based chemosensor exhibits reversible binding to the Hg²⁺ ion in the presence of other metal ions showing a 252-fold fluorescence enhancement and has potential applications as an INHIBIT logic gate and for cell-imaging.

Full text of DOI:10.1002/ejic.201300809

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DOI:10.1002/ejic.201300809
Rhodamine-Based Chromo-/Fluorogenic Dual Signalling
Probe for Selective Recognition of HgII with Potential
Applications for INHIBIT Logic Devices and Cell-
Imaging Studies
Tarun Mistri,[a] Rabiul Alam,[a] Malay Dolai,[a]
Sushil Kumar Mandal,[b] Pratik Guha,[a]
Anisur Rahman Khuda-Bukhsh,[b] and Mahammad Ali*[a]
Keywords: Mercury / Sensors / Logic gates / Cell-imaging / Fluorescence
A new rhodamine-based dual signaling probe (L3) has been
found to display quick responses through visible colorimetric
changes as well as fluorogenic properties on selective 1:1
binding to Hg2+, as delineated by absorption and fluores-
cence titrations as well as by JobЈs method and ESI-MS+
studies. The fluorescent probe L3 displays a 252-fold fluores-
cence enhancement on binding to Hg2+. A Benesi–Hilder-
its solubility in mixed organic/aqueous solvents as well as its
cell permeability, L3 could be used for in vitro/in vivo cell
imaging of Hg2+ with no or negligible cytotoxicity. The detec-
tion limit of Hg2+ was calculated by the 3σ method to be
9.28 ngL–1. Moreover, the fluorescence of the L3–Hg2+ sys-
tem was turned off and its red colour disappeared when KI
was added. Thus, the “OFF–ON–OFF” fluorescence sensing
behaviour of L3 observed in the presence of Hg2+ and I– may
find applications in devices with logic gate functions.
band fit of the absorption titration data gives Kd
=
(32.01Ϯ0.74) μM and a 1:1 binding stoichiometry. Owing to
ment of a wide variety of diseases, such as prenatal brain
damage, serious cognitive and motion disorders, and
Minamata disease.[3] Owing to the concern over its
deleterious effects in humans, new mercury detection meth-
ods have been developed that are cost effective, rapid, facile
and applicable in the environmental and biological
milieux.[4]
The most common analytical methods used for the detec-
tion and quantification of mercury ions include atomic ab-
sorption and emission spectroscopy,[5] inductively coupled
plasma mass spectroscopy (ICP-MS),[6] inductively coupled
plasma atomic emission spectrometry (ICP-AES)[7] and vol-
tammetry.[8] However, most of these methods are either very
costly or time-consuming and not suitable for performing
assays. Thus, over the past decade more attention has been
focused on the development of efficient fluorescent chemo-
sensors for the detection of Hg2+ ions for real-time moni-
toring of environmental, biological and industrial sam-
ples.[4,9,10]
The sensing chemistry of the Hg2+ ion is quite interest-
ing. It is known to be a fluorescence quencher mainly due
to its high atomic number (Z) and large spin–orbit coupling
(ζ)[11] but, in general, any optical feedback that translates
the metal-ion–receptor binding phenomenon into a fluores-
cence turn-on (fluorescence enhancement) response is pre-
ferred over the fluorescence turn-off (fluorescence quench-
ing) response process because quenching is not as sensitive
as enhancement and, moreover, fluorescence “turn-off”
Introduction
In recent years, the field of chemosensing has expanded
very rapidly due to its advantages of quick response, good
selectivity and high sensitivity and finds wide applications
in various measurement devices in the fields of life sciences,
environmental science, medical diagnostics and toxicologi-
cal analysis.[1] In particular, it is highly demanding for the
selective sensing of heavy metal ions such as Hg2+, Pb2+
and Cd2+ [2]
.
Mercury has significant industrial and agricultural uses
although it is known to be one of the most toxic heavy
metals. It is widespread in air, water and soil, generated by
many processes such as oceanic and volcanic eruptions, coal
burning, combustion of fossil fuels, gold mining and solid-
waste incineration. Mercury ions show a high affinity for
thiol groups in proteins and enzymes, leading to the mal-
function of cells and consequently causing health problems
in the brain, kidney and central nervous system. Its ac-
cumulation in the body can be responsible for the develop-
[a] Department of Chemistry, Jadavpur University,
Kolkata 700032, India
E-mail: m_ali2062@yahoo.com
http://www.jaduniv.edu.in/profile.php?uid = 30
[b] Department of Zoology, Kalyani University,
Kalyani 741235, India
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201300809.
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probes may produce false positive reports caused by other atoms for the selective and rapid recognition of toxic Hg2+
quenchers present in practical samples. Thus, any sensor ions that exhibits reversible fluorescence enhancement
molecule that shows a fluorescence turn-on response and along with a colour change visible to the naked eye through
can also be used for the colorimetric detection of Hg2+ in metal-induced opening of the spiro-lactam ring in the xan-
aqueous or mixed aqueous/organic solvent systems is im- thene moiety in an organic/aqueous medium with potential
portant from the viewpoint of its potential applications as for biological cell imaging.[17] Also, this study has revealed
a staining or imaging reagent for biological applications.[12] new potential applications in the development of new-gen-
The rhodamine framework is an ideal model for the con- eration molecular electronic devices, a molecular INHIBIT
struction of OFF–ON fluorescent chemosensors due to its logic gate, in which Hg2+ and I– are used as chemical inputs
particular structural properties.[13] Its derivatives with the and the fluorescence signal as output.
spiro-lactam structure are found to be non-fluorescent,
whereas opening of the spiro-lactam ring gives rise to a
strong fluorescence emission along with a drastic colour
change visible to the naked eye. Moreover, the rhodamine
fluorophore exhibits good photostability, a high extinction
coefficient and a longer emission wavelength (Ͼ550 nm),
Results and Discussion
Sensor L3 was easily synthesized by simple Schiff-base
condensation between rhodamine 6G hydrazide (L1) and 2-
(pyridin-2-ylmethoxy)benzaldehyde (L2) in methanol
which is often preferred to avoid background fluorescence
(Scheme 1) and thoroughly characterized by ESI-MS+, H
and 13C NMR spectroscopy, and CHN elemental analysis.
The structure of the probe was also analysed by single-crys-
tal X-ray diffraction (see Table S1 in the Supporting Infor-
mation) and the ORTEP view is shown in Figure 1. The
characteristic peak at δ = 64.79 ppm in the 13C NMR spec-
trum corresponding to C4 in L3 suggests that it exists in
solution predominantly in its colourless and fluorescence-
inactive spiro-lactam form.[18] The crystal structure of L3
also clearly shows the unique formation of the spiro-lactam
ring.
1
(below 500 nm).[13,14] It is also known to be an excellent
candidate as a fluorescent sensor that can detect Hg2+ ions
below 2 ppb.[13d] Thus, it provides both in-field detection
through optical colour change as well as the edge in im-
aging studies through its OFF–ON fluorescent property.
Because of all the above-mentioned advantages, rhodamine-
based fluorosensors have become an attractive choice for
the design of receptors for the Hg2+ ion.
On the other hand, remarkable progress has been
achieved in recent times in the development of molecular
systems that are capable of performing logic operations and
are attractive for the construction of molecular devices that
are demanded by information technology.[15] Also, their in-
tegration into arithmetic systems has brought chemists
closer to the realization of a molecular-scale calculator (mo-
leculator).[15c] Logic systems consisting of chemically en-
coded information as input and a fluorescence signal as
output have received considerable attention and functions
such as AND, NAND, OR, NOR, XOR, XNOR and
INHIBIT have been widely explored.[16] In all molecular
logic gates, the molecule demonstrates “ON” or “OFF”
switching of the fluorescence signal, which equates to an
output “1” or “0”, respectively, in response to the addition
“1” or no addition “0” of the input chemicals. Such devices
operate in wireless mode and have the potential for compu-
tation on a molecular level that silicon-based devices cannot
address. Hence, they have possible applications in the devel-
opment of electronic and photonic devices.
The spectrophotometric titration performed to analyse
the interaction of L3 with Hg2+ at 25 °C in aqueous MeCN
(4:6, v/v, HEPES buffer, pH 7.1) revealed that there is a
gradual development of a new absorption band centred at
around 529 nm on gradual addition of Hg2+ in the range
0–100.0 μm (Figure 2, A) and it becomes saturated upon ad-
dition of around 1.20 equiv. of Hg2+ (Figure 2, B), the con-
centration of L3 being fixed at 20.0 μm. A detectable change
in the colour of the solution from colourless to orange-yel-
low (Figure 2, E) clearly demonstrated the formation of the
ring-opened amide form of L3 upon binding to Hg2+ [19]
The composition and binding constant of the formed com-
plex [Hg(L3)] was determined by utilizing the Benesi–
Hilderband equation (1)
.
Therefore there is a need to develop new synthetic strate-
gies, usually employing a structurally well-developed recep-
tor unit in the fluoro-ionophore system that provides a re-
(1)
versible colorimetric as well as a fluorescent turn-on re- in which A0 and Amax are the absorbances of the pure
sponse essential for investigating intracellular Hg2+ ions ligand in the absence of the metal ion and in the presence
with no or negligible cytotoxicity. In particular, such fluo- of excess metal ion, respectively. A plot of [(Amax – A0)/
rescent chemosensors can be used not only for the selective (A – Ao)] vs. 1/[Hg2+] (Figure 2, C) gives a straight line with
detection of Hg2+ as a representative of heavy metal ions in Kd = (32.1Ϯ0.74) μm. It also indicates a 1:1 (n = 1) com-
organic/aqueous solutions and cells, but also for integrating plexation between L3 and Hg2+. The composition of the
these heavy metal ions in chemical-driven molecular ma- complex was also determined by Job’s method (Figure 2D)
chines in future molecular computing.
and is supported by mass spectrometric analysis (m/z =
We present herein a new type of easily synthesizable rho- 849.29 [Hg(L3)(H2O)Li]+; see Figure S1 in the Supporting
damine-based chemosensor with potential N2O2 donor Information).
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Scheme 1. Synthetic routes to chemosensor L3 and its binding mode towards Hg2+
.
Figure 1. ORTEP view of ligand L3 with ellipsoids drawn at the
30% probability level. Symmetry code: a = x, y, z; b = –x, 1/2 + y,
1/2 – z; c = –x, –y, –z; d = x, 1/2 – y, 1/2 + z.
The emission spectra of L3 and its fluorescence titration
with Hg2+ were also performed in MeCN/water solution
Figure 2. (A) Change in the absorption spectra of L3 (20 μm) upon
addition of Hg2+ in HEPES buffer at pH 7.1 in H2O/MeCN (4:6,
v/v) at 25 °C with [Hg2+] = 0–100 μm. (B) Plot of the absorbance
at 529 nm vs. [Hg2+]. (C) Benesi–Hilderband plot of (Amax – A0)/
(A – A0) vs. 1/[Hg2+]. (D) Job’s plot. (E) Photographs of solutions
of L3 and the L3–Hg2+ complex in MeCN/water.
(6:4, v/v, HEPES buffer, pH 7.1; Figure 3A). On gradual
addition of Hg2+ (0–35 μm) to the non-fluorescent solution
of L3 (7 μm), a 252-fold enhancement in fluorescence inten-
sity at 553 nm was observed following excitation at 508 nm,
which also suggests the opening of the spiro-lactam ring in
L3 on coordination to the Hg+2 ion.[19] The non-linear least-
squares fit of the fluorescence titration data of L3 (7.0 μm)
with Hg2+ (0–35 μm) gives an association constant K of
(6.48Ϯ1.84)ϫ105 m–1 (see Figure S2 in the Supporting In- colour or spectral change under identical conditions (Fig-
formation).
ure 4, A,B). For practical applications, the appropriate pH
The detection of Hg2+ was not perturbed by the presence conditions for successful operation of the sensor were evalu-
of biologically abundant Na+, K+, Ca2+ and Mg2+ ions. ated. No obvious fluorescence emission of L3 was observed
Similarly, several transition-metal ions, namely Cr3+, Mn2+
,
between pH 5 and 12, which suggests that the spiro-lactam
Fe2+, Fe3+, Co2+, Cu2+, Ni2+ and Zn2+, and heavy-metal form of L3 is stable over this wide range of pH and can
ions, like Cd2+, Pb2+ and Ag+, did not show any significant work well under physiological pH conditions (Figure 3, B).
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Figure 3. (A) Change in the fluorescence intensity of L3 (7 μm) upon addition of Hg2+ in HEPES buffer at pH 7.1 in H2O/MeCN (4:6,
v/v) at 25 °C with [Hg2+] = 0–35 μm (λex = 508 nm); inset: plot of fluorescence intensity (at 553 nm) vs. [Hg2+]. (B) pH–stability study of
the sensor L3 (6 μm); inset: fluorescence intensity vs. pH plot at 556 nm.
Figure 4. (A) UV/Vis spectral study of the selective binding of L3 (10 μm) towards Hg2+ over other metal ions (5 equiv.). (B) Fluorescence
study of the selective binding of L3 (7 μm) towards Hg2+ over other metal ions (5 equiv.).
A short response time and reversible response are also mined by the 3σ method[20] and found to be as low as
necessary for a fluorescent sensor to monitor Hg2+ ions in 9.28 ngL–1. All these findings indicate that L3 behaves as a
practical applications. The time dependence of the response good example of an ideal chemosensor for Hg2+ recogni-
of L3 to the Hg2+ ion was investigated and the results reveal tion.
that the recognition event could occur almost immediately
The mechanistic pathway proposed for the formation of
upon mixing (Յ1 min; see Figure S3 in the Supporting In- the L3–Hg2+ complex by opening of the spiro-lactam ring
formation). The reversible binding of L3 to Hg2+ was inves- was established through IR and H and 13C NMR studies.
1
tigated in the same solvent system as used in the absorption The IR study revealed that the characteristic stretching fre-
and fluorescence titrations (see Figure S4 in the Supporting quency of the amidic “C=O” of the rhodamine moiety at
2–
Information). Various anions, for example, SO42–, SO3
,
1710 cm–1 is shifted to a lower wavenumber (1649 cm–1) in
–
–
NO3 , PO43–, CO32–, S2–, Cl–, F–, Br–, I–, OAc– and N3 , the presence of 1.2 equiv. of Hg2+ (Figure 5). This large
were introduced into the solution containing L3 and Hg2+ shift signifies a strong polarization of the C=O bond upon
and the changes in their absorption and fluorescence inten- efficient binding to the Hg2+ ion and in fact indicates cleav-
1
sities were monitored after 3 min. The results show that age of this bond. Also, the H NMR spectra show the ring
anions like I– and S2– have a strong affinity towards Hg2+ protons (f and g; see the labelling in Figure S5 in the Sup-
and their binding constants may be much higher than that porting Information) of the rhodamine moiety are shifted
of L3. This causes demetallation of Hg2+ from the complex downfield in the presence of 1.2 equiv. of Hg2+ ions. The
and regeneration of the spiro-lactam ring with bleaching of “p” proton shows an upfield shift mainly due to an increase
the absorption band at 529 nm and the emission band at in electron density arising from the opening of the spiro-
around 553 nm. The quantum yield of the L3–Hg2+ com- lactam ring. The complex signal pattern of the other aro-
plex was determined to be Φ = 0.783 (with rhodamine 6G matic protons also indicates the involvement of the pyridine
used as a standard), whereas the free ligand is non- or very moiety of the receptor unit of L3 in the binding of Hg2+
.
weakly fluorescent with a very negligible absorption band Also, the disappearance from the 13C NMR spectrum of
at 529 nm. The limit of detection (LOD) of Hg2+ was deter- the signal at δ = 64.79 ppm for the tertiary carbon (sp3-
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hybridized) of the spiro-lactam ring of L3 (labelled as C4, probe acts in parallel with the fluorescence output signals,
see Figure 6) upon addition of 1.2 equiv. of Hg2+ convinc- which implements the required AND function. The combi-
ingly supports the opening of the spiro-lactam ring and co- nation of both inputs leads to fluorescence quenching as
ordination through the O atom of the -CONH group of the output 0, in accordance with the Truth Table in Figure 7
rhodamine moiety to the Hg2+ ion.[21]
(B). Therefore monitoring the fluorescence at 553 nm upon
addition of Hg2+, I– and a mixture of the two satisfies an
INHIBIT logic gate function (Figure 7).
Figure 5. IR spectra of L3 and L3–Hg2+ in CH3CN.
The INHIBIT logic gate involves a particular combina-
tion of the LOGIC functions AND and NOT. For our sys-
tem we can make a correlation by taking two input signals,
namely input 1 (Hg2+) and input 2 (I–), along with fluores-
cence signal of the probe L3 (5 μm) at 553 nm as the output.
For the input, the presence and absence of Hg2+ and I–
are assigned as 1 (ON state) and 0 (OFF state), respectively.
For output, we assign the enhanced fluorescence of L3 as 1
Figure 7. (A) Output signals (at 553 nm) of the logic gate in the
presence of different inputs. (B) Corresponding Truth Table of the
logic gate. (C) General representation of an INHIBIT gate.
(D) UV-exposed photographs of L3, L3–Hg2+ and a mixture of L3–
Hg2+ + I–in MeCN/H2O (6:4, v/v, pH 7.1, HEPES buffer) solvent.
The intracellular Hg2+ imaging behaviour of L3 was
(ON state) and the quenched fluorescence as 0 (OFF state; studied by using HeLa cells in conjunction with fluores-
see the Truth Table in Figure 7, B). In the absence of both cence microscopy. After incubation with L3, the cells dis-
inputs (Hg2+ or I–), L3 remains in the OFF-state form. In- played no intracellular fluorescence (Figure 8, B). However,
put 1 leads to significant fluorescence enhancement through the cells exhibited intense fluorescence when a solution of
its interaction with the free receptor in its occupied state Hg(ClO4)2 (10–50 μm) was added to the L3 pre-incubated
leading to the ON-state form, but the interaction of input cells (Figure 8, C–E). However, the intense fluorescence was
2 with its corresponding receptor leads to the OFF-state strongly suppressed when KI (100 μm) was also added to
form, thereby implementing the necessary NOT gate. The this medium. Because KI shows a strong scavenging action
Figure 6. 13C NMR spectra of L3 and L3–Hg2+ in [D6]dmso.
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on Hg2+, the sensor was competitively inhibited from bind- tial applications of the L3–Hg2+ system as a device with
ing to Hg2+, and so the fluorescence almost disappeared logic gate functions and therefore shows advantages for the
(Figure 8, F). This confirms the ability of the L3 to sense development of new-generation digital devices.
Hg2+ ions in biological systems under physiological condi-
tions. Also, L3 can effectively be used to monitor changes in
the intracellular concentration of Hg2+ ions under different
biological conditions, particularly because of its relatively
low cytotoxicity up to 8 h (Figure 9).
Experimental Section
Materials and Instruments: Steady-state fluorescence studies were
carried out with a Shimadzu (Model RF-5301PC) and Horiba–
Jobin–Yvon Fluoromax-3 spectrofluorimeters. UV/Vis absorption
spectra were recorded with an Agilent 8453 diode array spectro-
photometer. NMR spectra were recorded with a Bruker spectrome-
ter at 300 MHz. The ESI-MS+ spectra were recorded with a Qtof
Micro YA263 mass spectrometer.
All solvents used for synthesis were of reagent-grade (Merck) un-
less otherwise mentioned. For spectroscopic (UV/Vis and fluores-
cence) studies HPLC-grade MeCN and double-distilled water were
used. Rhodamine 6G hydrochloride, 2-chloromethylpyridine and
metal salts such as the perchlorates of Na+, K+, Ca2+, Fe2+, Co2+
,
Ni2+, Zn2+, Pb2+, Cd2+, Hg2+ and Cu(NO3)2·2H2O were purchased
from Sigma–Aldrich and used as received. All other compounds
were purchased from commercial sources and used as received.
Figure 8. (A) Phase contrast. (B) Fluorescence image of HeLa cells
in the absence of chemosensor L3. Chemosensor L3 (10 μm)-incu-
bated cells washed with PBS (pH 7.2) and exposed to increasing
concentrations of extracellular Hg2+ ions: (C) 10, (D) 20 and
(E) 50 μm. (F) Image E + KI (100 μm). For all imaging, the samples
were excited at 508 nm.
Preparation of Rhodamine 6G Hydrazide (L1): Rhodamine 6G
hydrazide was prepared according to a literature method.[22]
Preparation 2-(Pyridin-2-ylmethoxy)benzaldehyde (L2): L2 was pre-
pared by a modification of a literature procedure.[23] Salicylalde-
hyde (10 mmol, 1.23 g) and K2CO3 (18 mmol, 2.52 g) were added
to dry MeCN (60 mL), and the mixture was heated at reflux for
40 min. 2-Picolyl chloride (11.5 mmol, 1.89 g) and a catalytic
amount of KI (0.15 g) was then added to the above reaction mix-
ture, which was heated at reflux for a further 12 h. Then the mix-
ture was cooled and filtered. The filtrate was evaporated to one
third of its initial volume and diluted with water (40 mL). Then the
pH of the solution was adjusted to 4 by the addition of 1 m HCl
and extracted with dichloromethane (dcm; 2ϫ40 mL). The pH of
the aqueous solution was then adjusted to 8 by the addition of
4.0 m Na2CO3 solution and extracted with dcm (3ϫ40 mL). Then
the combined organic phase after drying with anhydrous Na2SO4
was evaporated to dryness under reduced pressure to give a pale-
yellow solid residue. The crude solid product was recrystallized in
MeOH/dcm (8:2, v/v) to give the desired NMR-pure product as
Figure 9. Cell viability (determined by the MTT assay) of HeLa
cells treated with different concentrations of chemosensor L3 for
8 h.
1
an off-white crystalline solid (66% yield), m.p. 69.4 °C. H NMR
Conclusions
(CDCl3): δ = 10.62 (s, 1 H), 8.62 (dd, 1 H), 7.76 (td, 1 H), 7.54 (m,
2-H), 7.29 (ddd, 1 H), 7.07 (m, 2 H),5.33 (s, 2 H) ppm. 13C NMR
(CDCl3): δ = 189.53, 160.59, 156.32, 149.35, 137.04, 135.98, 128.85,
We have designed and synthesized a new rhodamine-
based chromo-/fluorogenic dual signalling probe for selec-
tive recognition of Hg2+, which has been characterized by
1H and 13C NMR, IR, ESI-MS and single-crystal X-ray
diffraction. The rhodamine-based ligand L3 displays visible
colorimetric as well as fluorogenic properties on selective
binding to Hg2+ with a 252-fold enhancement of the
125.16, 123.01, 121.26, 121.21, 113.04, 71.10 ppm. IR: ν = 1690
˜
(CHO), 1253 (aliphatic C–O) cm–1. MS (ES+): m/z = 214.07 [L +
H]+. C13H11NO2 (213.24): calcd. C 73.19, H 5.21, N 6.62; found C
73.37, H 5.09, N 6.58.
Preparation of Probe L3: L2 (1.1 mmol, 0.234 g) in MeOH (10 mL)
fluorescence. Benesi–Hilderband analysis of the absorption was added dropwise over 30 min to a methanolic solution (30 mL)
of L1 (1 mmol, 0.428 g) containing 1 drop of acetic acid under hot
titration data gives Kd = (32.01Ϯ0.74) μm with 1:1 com-
(ca. 50–60 °C) conditions. Then the reaction mixture was stirred
plexation. Owing to its solubility in MeCN/H2O and cell
for around 6 h at room temperature. An off-white precipitate was
permeability it could be used for in vitro/in vivo cell-im-
formed, which was collected by filtration. The residue was washed
aging of Hg2+ with no or negligible cytotoxicity. The detec-
thoroughly with cold methanol to isolate L3 in pure form in 84%
tion limit of Hg2+ was calculated by the 3σ method to be
yield. Single crystals of L3 were obtained by recrystallization in
9.28 ngL–1. Moreover, reversible binding of L3 to Hg2+ may
be useful in real-time applications, which is superior to
irreversible binding of a probe towards a metal ion. Thus,
the “OFF–ON–OFF” fluorescence sensing behaviour ob-
1
dcm/MeCN (1:1, v/v), m.p. 222.2 °C. H NMR (CDCl3): δ = 8.76
(s, 1 H), 8.57 (s, 1 H), 7.99–8.05 (m, 2 H), 7.80 (t, 1 H), 7.46 (t, 2-
H), 7.22–7.26 (s, 2-H), 7.16 (t, 1-H), 7.06 (t, 1 H), 6.90 (t, 1 H),
6.74 (d, 1 H), 6.39 (s, 2 H), 6.30 (s, 2 H), 5.11 (s, 1 H), 3.43 (br., 2
served in the presence of Hg2+ and I– strengthens the poten- H), 3.11 (q, 4 H),1.87 (s, 6 H), 1.25 (t, 6 H) ppm. 13CNMR ([D6]-
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dmso): δ = 163.99, 156.3, 152.32, 150.5, 148.94, 147.7, 139.3, 137.0,
134.0, 127.7, 126.5, 124.9, 123.0, 122.9, 121.5, 121.04, 118.3,
113.44, 104.44, 95.88, 70.47, 64.79, 37.5, 16.94, 14.06 ppm. MS
University Grants Commission (UGC), New Delhi (UGC-NET).
S. K. M. is grateful to the DST for partial financial support
through the PURSE Program.
(ES+): m/z = 624.22 [L + H]+. IR: ν = 1710 (spiro-lactam amide
˜
keto), 1621 (–C=N), 1261 (aliphatic C–O) cm–1. C39H37N5O3
(623.75): calcd. C 75.11, H 5.99, N 11.23; found C 74.98, H 5.85,
N 11.76.
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Cell Imaging Experiment and Cytotoxicity Studies
Cell Culture: The HeLa cell line of human cervical cancer origin
was obtained from the National Center for Cell Science, Pune, In-
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1% antibiotic mixture containing penicillin, streptomycin and neo-
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5% CO2. The cells were grown to 80–90% confluence, harvested
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in phosphate-buffered saline (PBS), plated at the desired cell con-
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Cell-Imaging Study: Cells were rinsed with PBS and incubated with
DMEM containing L3 making the final concentration up to 10 μm
in DMEM; the stock solution (1 mm) was prepared by dissolving
L3 in the mixed solvent (dmso/water = 1:9, v/v) for 30 min at 37 °C.
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an objective lens of 20ϫ magnification. Fluorescence images were
taken of the HeLa cells incubated with 10 μm L3 for 30 min and
after the addition of different concentrations (10–50 μm) of Hg2+
ions and KI (100 μm).
Cell Cytotoxicity Assay: To test the cytotoxicity of L3, the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as-
say was performed following the procedure described previously.[24]
After treatment with 1, 10, 20, 50 and 100 μm L3 for 8 h, 10 μL of
MTT solution (10 mg/mL PBS) was added to each well of a 96-
well culture plate and again incubated at 37 °C for a period of 3 h.
All media were removed from the wells and acidic isopropyl alcohol
(100 μL) was added to each well. The intracellular formazan crys-
tals (blue-violet) formed were solubilized with 0.04 n acidic iso-
propyl alcohol and the absorbance of the solution was measured
at 595 nm with a Thermo Multi Scan EX microplate reader. The
cell viability was expressed as the optical density ratio of the treated
sample and control. Values are expressed as the mean of three inde-
pendent experiments. The cell cytotoxicity was calculated as the %
cell cytotoxicity = 100% – % cell viability.
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Acknowledgments
Financial support from the Department of Science and Technology
(DST), Government of India, New Delhi (ref. number SR/S1/IC-
20/2012) and the Council of Scientific and Industrial Research
(CSIR), New Delhi [ref. number 01(2490)/11/EMR-II] is gratefully
acknowledged. T. M. and M. D. acknowledge fellowships from the
Eur. J. Inorg. Chem. 2013, 5854–5861
5860
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Published Online: October 8, 2013
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5861
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