X-ray structurally characterized sensors for ratiometric detection of Zn2+ and Al3+ in human breast cancer cells (MCF7): development of a binary logic gate as a molecular switch

Article abstract of DOI:10.1039/c5dt01303h

A very simple molecule derived from salicylaldehyde and N-phenyl ethylenediamine (L1) functions as dual-mode ratiometric fluorescence "turn on" sensors for Zn²⁺ and Al³⁺ at two different wavelengths. The sensing is based on the combi

Full text of DOI:10.1039/c5dt01303h

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DOI: 10.1039/C5DT01303H
ARTICLE
X-ray structurally characterized sensors for
ratiometric detection of Zn2+and Al3+ in human breast
cancer cell (MCF7): Development of binary logic gate
as molecular switch
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
Abhijit Ghosh and Debasis Das*
DOI: 10.1039/x0xx00000x
Department of Chemistry, The University of Burdwan, Golapbag, Burdwan, India, Fax:
+91-342-2530452; Tel: +91-342-2533913 (ext. 424); E-mail: ddas100in@yahoo.com
www.rsc.org/
A very simple molecule derived from salisaldehyde and N-phenyl ethylenediamine (L1) functions as
dual-mode ratiometric fluorescence “turn on” sensors for Zn2+ and Al3+ at two different wavelengths.
The sensing is based on the combined effect of inhibition of excited-state intra-molecular proton transfer
(ESIPT), CH=N isomerization and chelation-enhanced fluorescence (CHEF). Moreover, [L1+Zn2+]
system functions as better Al3+ sensor where Al3+ ratiometrically displaces Zn2+ from [L1+Zn2+]
complex. Emission wavelength dependent differentiation of Zn2+ and Al3+ using L1 allows us to develop
a binary logic gate that functions as a molecular switch. L1 efficiently detects Zn2+ and Al3+ in human
breast cancer cell (MCF7) while [L1+Zn2+] complex specifically detects Al3+ in the said cells.
Chemosensors that convert molecular recognition event into processes.16 However, its excessive exposure (WHO recommended
highly sensitive and easily detectable signals are being actively average daily human intake of aluminium is approximately 3–10
studied as an attractive research area indeed. The imaging techniques mg17) may cause severe toxicity on human’s health, particularly
to visualize bio-active metal ions have got superior importance in neurotoxicity to damage central nervous system. Disorders of
biomedical analysis and environmental monitoring. Fluorescence aluminium homeostasis lead to a number of diseases, such as
technique has emerged as most powerful tool for its operational Parkinson’s disease (PD), Alzheimer’s disease (AD) and
simplicity, high sensitivity and cost effective methodology.1,2
amyotrophic lateral sclerosis.18-21 In addition, Al3+ is suspected to be
Particularly, when ratiometric sensor exhibits spectral shift upon involved in disease like microcytic hypochromic anemia, myopathy,
interaction with specific analyte, then combination of fluorescence and affects the absorption of iron in blood causing anemia.22,23
sensing and imaging allow its’ most promising and vibrant method Moreover, it inhibits plant growth on acid soils.24
of determination. A ratiometric sensor is highly desirable as it
Thus, due to the potential impact of Zn2+ and Al3+ levels on human
reduces or eliminates the adverse effects of change of environment health and environment, selective trace level detection of both the
around the probe (pH, polarity, temperature etc.), probe ions in a single shot is highly demanding.
concentration and excitation power on the emission intensity by
providing a built-in correction to the signal ratio of emission (input) into optical signal as output is the bases of molecular logic
intensities at two different wavelengths.3-5 gates,25-28 a potential scientific interest in the field of unconventional
Zn2+ plays pivotal roles in numerous biological processes including computing systems.29 Examples include AND, OR, XOR, INHIBIT
brain activity, gene transcription, and immune function etc.6 It is and NAND type molecular level logic gates.30-32
Molecular arithmetic to convert chemically encoded information
.
employed as a required cofactor that can stimulate the activities of
Till date, very few fluorescence sensors33-38 can selectively detect
approximately 300 enzymes7 including those involved in gene both Zn2+ and Al3+. Additionally, having similar chemical properties,
expression and DNA repair.8,9 However, abnormal zinc metabolism Cd2+ interferes with the Zn2+ sensor.39
is associated with many health problems, such as prostate cancer,
From practical point of view, a single sensor that shows
delayed sexual maturation and impotence, type 2 diabetes mellitus differential response towards multiple ions is more desirable than
(T2DM), Wilson’s diseases, amyotropic lateral scelerosis (ALS) and one-to-one sensor. These facts stimulated us to search for a single
age-related macular degeneration (AMD).7,10,11 Plausibly, Zn2+ probe that can detect both Zn2+ and Al3+ selectively.
homeostasis have some correlation on the pathology of Alzheimer’s
Recently, our research group has developed a new turn-on Zn2+
disease12 and other severe neurological problems such as cerebral and Al3+ dual sensor40. However that is not also the most desirable
ischemia13 and epilepsy.14
ratiometric one as already discussed. Ratiometric sensor for dual
On the other hand, aluminium enters human body through foods sensing of Zn2+ and Al3+ is very rare. A single report from Goswami
and water as Al3+, trace amount of which is found in most biological group41 have several limitations, viz. the difference between their
tissues15 and required for various normal human physiological emission wavelengths is very narrow (14 nm, λem for Zn2+, 446 nm
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and λem for Al3+ is 432 nm). Moreover, the ratiometric behaviour is Zn2+ and 415 nm for Al3+. The plots of emission intensity of L1 as a
not so prominent/ distinct, while the logic gates constructed are not function of added Zn2+ and Al3+ are presented in Fig.S3 and S4 (ESI)
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satisfactory/ useful. Moreover, structures of those probes have not respectively. The linear regions of the plots (Fig.S1a and S2a, ESI)
2+
3+
DOI: 10.1039/C5DT01303H
been authenticated by most reliable single crystal X-ray analysis.The are useful for determination of unknown Zn
same group have recently reported42 dual channel detection of Zn2+ concentrations. In presence of Zn2++, 44-fold enhancement of F450 340
and Al3+ that too has several limitations viz. complex and multi-step (0.03277 to 1.43164) has been achieved (Fig.S5, ESI), whereas 100-
synthetic protocol with significantly low yield of the probe and used fold enhancement of F485 340
(0.01799 to 1.8112) is observed for Al3+
and Al
/
/
acetonitrile, a very toxic solvent as medium. Moreover, the structure (Fig.S6, ESI). Fluorescence titration profiles provide the association
of the Zn2+complex is not authenticated by single crystal X-ray constants of L1 for Zn2+ and Al3+ as 4.13 × 105 M─1 and 2.04 × 106
structure analysis.
M─1 following Hill plots43 (Fig.S7 and S8, ESI). The detection limits
Addressing those lacunas, we have been able to overcome those for Zn2+ and Al3+, estimated from fluorescence titration are 2.3 ×
limitations and, herein present a molecular probe, L1 capable of 10─7 M (Fig.S9, ESI) and 2.4 × 10──7 M (Fig.S10, ESI) respectively.
dual-sensing Zn2+ and Al3+ in a ratiometric manner in aqueous-
ethanol media with improved detection limits. The sensitivity of L1
towards Zn2+ and Al3+ is based on inhibition of both ESIPT and
CH═N isomerization along with occurrence of CHEF. Additionally,
the [L1-Zn2+] complex can selectively detect Al3+ under identical
experimental conditions. Much lower detection limit with practically
no interference have been observed for sensing of Al3+ by [L1-Zn2+]
complex compared to free organic probe, L1. Furthermore,
structures of both L1 and [L1-Zn2+] complex have been confirmed
by single crystal X-ray structure analysis. The probe L1 is useful to
image both Zn2+ and Al3+ while the [L1-Zn2+] complex selectively
images Al3+ in human breast-cancer cells (MCF7) under
fluorescence microscope. Experimental findings have been
substantiated by theoretical studies too.
Fig.1 Relative emission intensities of L1 (30 μM) in presence of different
metal ions (500 μM) in HEPES buffer (0.1 M, ethanol/ water = 1/1, v/v, pH
7.4) medium, λex, 276 nm
Scheme 1
Results and discussion
Spectral studies
Since L1 contains pH sensitive donor sites, its performance is pH
sensitive. Emission intensities of L1, both in presence and absence of
Zn2+ and Al3+ are examined at different pH. Fig.S1 and Fig.S2 (ESI)
indicate that L1 senses both Zn2+ and Al3+ over a wide pH range
from 6.0 to 11.0. However optimum performance is observed at pH
4.0-8.0. Hence, pH 7.4, being closer to the physiological pH is
Fig.2 Changes in the fluorescence spectra of L1 (20 µM) in HEPES buffered
chosen for the entire studies. All spectral studies have been
(0.1 M) solution (ethanol /water =1:1, v/v, pH 7.4) upon gradual addition of
performed in 0.1 M HEPES buffered aqueous ethanol media
Zn2+ (0.5-1000 µM).λex = 276 nm, λem = 340 nm, 450 nm.
(ethanol/water, 1/1, v/v).
The selectivity of L1 towards various common metal ions are
examined by addition of 5.0 equiv. Na+, K+, Ca2+, Mg2+, Cr3+, Mn2+,
Fe3+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+, Pb2+, Zn2+ and Al3+ ions to
an aqueous ethanol (1:1, v/v) solution of L1. Fig.1 shows that free
L1 displays an emission band at 345 nm (λex, 276 nm). Except Zn2+
and Al3+, no other common metal ions induce any change to the
emission of free L1.
Gradual addition of Zn2+/ Al3+ (Fig.2/ 3) decreases emission
intensity at 345 nm (λex, 276 nm) along with the appearance of a new
band at 450 nm (blue emission for Zn2+) and 485 nm (green emission
for Al3+) which gradually enhances with increasing amount of
respective metal ions. The red shift of the emission band is
accompanied by 27 fold fluorescence enhancement for Zn2+ (λem
,
450 nm, Ф, 0.19, 24 fold enhancement). Similarly 57 fold
Fig.3 Fluorescence spectral changes of L1 (20 µM) in HEPES buffered (0.1
M) solution (ethanol/water =1:1 v/v, pH 7.4) upon gradual addition of Al3+
(0.5-1000 µM); λex = 276 nm, λem = 340 nm, 485 nm
fluorescence enhancement (λem
, 485 nm, Ф, 0.30, 29 fold
enhancement) is observed for Al3+. Moreover, these ratiometric
responses are associated with clear isosbestic points at 400 nm for
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The chelation of Zn2+/Al3+ with O, N donors of L1 increases the alone. It will be inconclusive from the output whether Al3+ alone or
rigidity of the molecular assembly by restricting the free rotation together with Zn2+ is present. Our system uses combination of OR
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around the azomethine functionality and eliminates the ESIPT and NOT gates along with a switch that describes the situation very
2+
3+
st
DOI: 10.1039/C5DT01303H
process. These two effects are jointly responsible for the observed clearly. When any of the two inputs (Zn /Al ) is high (1 three
fluorescence enhancement (Fig.4).
rows in the truth table), the output (at λmax = 450 nm or 485 nm) is
the result of only OR gate (OR1) and the switch is connected to ‘A’.
When both Zn2+ and Al3+ inputs are “1” then the output is due to Al3+
only, not for Zn2+ i.e., at λmax = 485 nm but not at 450 nm. In this
case (last row in the truth table) the switch is connected to ‘B’.
Fig.4 Proposed sensing mechanism of Zn2+ and Al3+ by L1 and Al3+
by [L1 –Zn2+] complex
Interestingly, having greater affinity and higher association
constant for L1, Al3+ easily replaces Zn2+ from [L1–Zn2+] complex to
form more stable [L1–Al3+] complex. Thus, [L1–Zn2+] complex
turns out as a better Al3+ sensor and functions via displacement
approach. Serial addition of Al3+ to a solution of the [L1–Zn2+]
complex (Fig.5) results red shift of 450 nm emission band (λex, 276
nm) to 485 nm along with fluorescence enhancement. This
competitive fluorescence titration allows with an improved detection
limit for Al3+, 3.3 × 10─8 M (Fig.S11, ESI). Most plausibly, higher
co-ordination number of Al3+ than Zn2+ (6> 4) imparts more rigidity
to the system (vide Fig.4), and minimizes its entropy leading to
further fluorescence enhancement.
Fig.5 Fluorescence spectral changes of [L1 –Zn2+] complex (50 µM) in
HEPES buffered (0.1 M) solution (ethanol/ water = 1:1 v/v, pH 7.4) upon
addition of Al3+ (0.4-1000 µM); λex = 276 nm
The effect of competing metal ions on the detection of Zn2+/Al3+
is examined to validate the selectivity of L1 for Zn2+/Al3+. Addition
of 5.0 equiv. Zn2+/Al3+ to L1 in presence of 5.0 equiv. other metal
ions shows no significant interferences (Fig.S12 and S13, ESI).
Some minor interference for determination of Al3+ is completely
eliminated employing [L1–Zn2+] complex as Al3+ sensor (Fig.S14,
ESI). Fig.S15 (ESI) presents the colour changes of L1 upon addition
of equimolar various cations under UV light which allows an
effective and efficient detection of Zn2+ and Al3+.
The absorption spectrum of L1 exhibits a band at 305 nm,
attributed to π−π* transition. Upon gradual addition of Zn2+/Al3+, the
intensity of this band decreases along with the appearance of new
bands at 363 nm (for Zn2+), 385 nm (for Al3+). Four well-defined
isosbestic points have been observed (Fig.6 and Fig.7). On the other
hand, addition of Al3+ to [L1–Zn2+] complex reveals that the
absorption band initially present at 363 nm decreases with the
appearance of a new band at 385 nm (Fig.8). This clearly indicates
the displacement of Zn2+ from [L1–Zn2+] complex by Al3+ to form a
thermodynamically favorable [L1–Al3+] complex.
Fig.6 Changes in absorbance of L1 (50 µM) upon gradual addition of Zn2+ (1-
3000 µM) in HEPES buffered (0.1 M) solution (ethanol /water =1:1 v/v, pH
7.4)
Thus, Zn2+ and Al3+ dependent shift of emission wavelengths and
selective displacement of Zn2+ by Al3+ from the [L1–Zn2+] complex
have allowed us to construct a binary logic gate useful in digital
electronics (Fig.9). For input, Zn2+/Al3+ defined as “1” state and the
absence of any as “0” state. There are two input signals, viz. input 1
(Al3+) and input 2 (Zn2+) whereas the output signals correspond to
the turn on emission at 485 nm (output 1 due to Al3+) and 450 nm
(output 2 due to Zn2+). The logic gates constructed by Goswami
group is not at all well-presented, because they have mentioned same
output when both Zn2+ and Al3+ are present together or Al3+ present
Fig.7 Changes in absorbance of L1(50 µM) upon gradual addition of Al3+ (1-
3000 µM) in HEPES buffered (0.1 M) solution (ethanol /water =1:1 v/v, pH
7.4)
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S1 (ESI). The crystal packing of [ZnL1] complex is shown in
Fig.S21 (ESI).
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DOI: 10.1039/C5DT01303H
Fig.8 Changes in absorbance of L1 (50 µM) upon gradual addition of Al3+ (1-
3000 µM) in HEPES buffered (0.1 M) solution (ethanol /water =1:1 v/v, pH
7.4)
Fig.9 The truth table and logic diagram for simultaneous monitoring of
Zn2+and Al3+ using L1
For better understanding of the solution state interaction between
1
L1 with Zn2+ and Al3+, H NMR titration experiments have been
Computational studies
carried out independently in CD3OD-D2O media (Fig. 10, Fig.S16
and S17, ESI). 1H NMR spectrum of L1 shows very weak signals of
N─H and phenolic O─H protons at 7.53 ppm and 10.06 ppm as
expected for their exchangeable character with the solvent protons.
Upon addition of 0.5 equiv. Zn2+ to L1, the phenolic O─H proton (at
10.06 ppm) disappears (Fig.S16, ESI). Also, the CH═N proton is
down-field shifted from 8.35 ppm to 8.37 ppm. On the other hand,
upon addition of 0.5 equiv. Al3+, the phenolic O─H protonis up-field
shifted to some extent from 10.06 to 10.03 ppm with enhanced
intensity while the CH═N proton is downfield shifted from 8.35
ppm to 9.27 ppm (Fig.S17, ESI). Moreover, the N─H signal (7.53
ppm for free L1) is downfield shifted to 7.60 ppm with enhanced
intensity. These facts suggest the coordination of nitrogen donor
sites to Al3+ rendering N─H protons non-exchangeable. Other
aromatic and aliphatic protons show slight changes for both cases.
The results altogether suggest that Al3+ is coordinated with the imine
nitrogen, hydroxyl oxygen and sp3 nitrogen donors while Zn2+ is
coordinated via imine nitrogen and hydroxyl oxygen sites of L1. No
further change of any proton signals have been observed upon
addition of excess metal ions (over 0.5 equiv.) confirming 2:1
complexation between L1 and Zn2+/Al3+. The same stoichiometry is
also supported by the Job’s plot (Fig.S18 and S19, ESI). Single
crystal X-ray structure of the [L1-Zn2+] complex (Fig.S20, ESI)
helps to conclude that solid state structure of the complex is retained
in solution too. Complexation to Al3+ causes relatively higher
downfield shifts of the protons of L1, indicating stronger binding
relative to Zn2+.
Molecular level interactions between L1 and Zn2+/ Al3+ have been
studied using density functional theory (DFT) employing B3LYP/ 6-
31G and Lanl2dZ basis set.45 The HOMO- LUMO energy gaps in
free L1, and its Zn2+/Al3+ adducts are 3.8262 eV, 3.4433 eV and
3.3190 eV respectively (Fig. S22, ESI). With the optimized
structures of the complexes, electronic transition energies are
calculated by TDDFT method in gas phase and methanol. For
methanol, the CPCM formalism is imposed which considers the
solvent as a polarisable continuum and do not include discrete
solvent molecules for solvation of the solute.46 Calculated transition
energies for the prominent absorption bands are shown in Table S2
(ESI). Calculated absorption peaks agree well with the
experimentally observed peaks. The energy-optimised structure of
[L1-Zn2+] complex shows distorted octahedral geometry as also
found in SC-XRD structure. Thus, very close agreements between
the theoretical and experimental findings have been observed.
Cell imaging
L1 is capable to detect intracellular Zn2+/ Al3+ in human breast
cancer cells, MCF7 under fluorescence microscope. Cells treated
with L1 (without Zn2+/Al3+) are used as control. Fig.11 indicates that
L1 is able to permeate cell membrane to stain Zn2+/ Al3+ without any
harm (cells remain alive even after several hours of exposure to 20
μM L1) making it useful to monitor Zn2+ or Al3+ accumulation in
biological systems. Fig.11 also supports the displacement of Zn2+ by
Al3+ from its adduct with L1.
Addition of 5.0 equiv. Al3+ to the system containing [L1 + 5.0
equiv. Zn2+] brings the O─H signal back to its position found for [L1
+ 5.0 equiv. Al3+] (Fig.10). Further, CH═N proton experiences the
desired down-field shift from 8.37 ppm to 9.27 ppm and the N─H
signal intensity enhances. The final spectrum looks exactly same to
that found after addition of 5.0 equiv. Al3+ to L1. All these support
stoichiometric displacement of Zn2+ from [L1–Zn2+] complex by
Al3+.
The Zn2+ in Fig.S20 adopts a pseudo-tetrahedral coordination,
where the tetrahedron is somewhat distorted with N(1)─Zn─O(3)
angle being 95.5(3)º and N(4)─Zn─O(1) angle being 96.9(4)º. The
N(1)─Zn─N(4) and O(1)─Zn─O(3) angles have opened up to
112.92(12)º and 110.78(11)º respectively. The Zn─N distance
ranging from 1.973(N1) A° to 2.019(N4) A°, while, the Zn─O
distance ranging from 1.918(O1) A° to 1.936(O3) A°, are considered
normal.44 The most significant geometric data are presented in Table
Fig.10 1H NMR spectral changes of L1 upon addition of Zn2+ and Al3+
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between L1 and Zn2+. FTIR (cm−1) (Fig.S31, ESI): υ(N─H 2º amine)
3294, υ(CH=N) 1600, υ(C─O phenyl) 1303. The absence of υ(O-H,
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Experimental
Apparatus and reagents
High-purity HEPES, salisaldehyde and N-phenyl ethylenediamine phenol) band further confirms deprotonation of L1 upon interaction
2+
are purchased from Sigma Aldrich (India). Zn2+ and Al3+ stock with Zn . Thermogram (Fig.S32, ESI) shows 6.098% weight loss in
solutions have been prepared using Zn(NO3)2.6H2O and Al(NO3)3. the temperature range, 40.0-260.7ºC corresponds to the loss of two
9H2O salts respectively. All other metal ions used are either their oxygen atoms, and 21.376% weight loss in the temperature range,
nitrate or chloride forms. Other analytical reagent grade chemicals 260.7-343.3ºC, corresponds to the rupture of one CH=N bond along
are used without further purification except when specified. Water with loss of aldehyde unit. Again, 11.690% weight loss in the
having a resistivity of 18.2 MΩ cm is obtained from a Milli-Q temperature range, 343.3-452.9ºC corresponds to the breaking of
Millipore water purification system (Bedford, MA) and used second CH=N bond with loss of corresponding aldehyde unit and
throughout all the experiments. Solvents used are of spectroscopic 43.530% weight loss in the temperature range, 452.9-566.1ºC is
grade. A Shimadzu Multi Spec 2450 spectrophotometer is used for attributed to the loss of two amine units. Thermogram also indicates
recording UV-Vis spectra. FTIR spectra are recorded on a Shimadzu that [L1+Zn2+] complex is more thermally stable (up to 260.7ºC)
FTIR (model IR Prestige 21 CE) spectrophotometer. Thermo over L1 (up to 200ºC). UV-Vis. (Fig.S33, ESI): λ (nm) in EtOH/
gravimetric analyses are performed on a Perkin Elmer TG/DTA lab H2O, 1/1, v/v (ε, M−1 cm−1): 363 nm (32000), 271 nm (75500).
system l (Technology by SII). Mass spectra have been performed Excitation spectrum using emission wavelength is presented in in
using a QTOF 60 Micro YA 263 mass spectrometer in ES positive Fig.S33, (ESI).
DOI: 10.1039/C5DT01303H
mode. 1H and 13C NMR spectrum are recorded using Bruker Avance
DPX 300 (300 MHz) in CDCl3. H NMR titration experiments have
1
Synthesis of [L1+Al3+] complex
been performed using Bruker Avance DPX 600 (600 MHz) in
A methanol solution of Al(NO3)3. 9H2O (50 mg, 0.1332 mmol) is
CD3OD-D2O system. Elemental analyses are performed on a Perkin added slowly to a magnetically stirred solution of L1 (64 mg, 0.2665
Elmer 2400 CHN analyzer. The steady state emission and excitation mmol) in methanol. Stirring is continued for 1h. On slow
spectra are recorded with a Hitachi F-4500 spectrofluorimeter. evaporation of the solvent, a deep yellow-brown compound is
Systronics digital pH meter (model 335) is used for pH obtained. Anal. calcd (%): C, 70.99; H, 6.35; N, 11.04; O, 6.30
measurement. Fluorescence microscope images are captured through found: C, 70.82; H, 6.49; N, 10.86; O, 6.17. The compound is
an Olympus IX81 microscope and processed using image-pro plus characterized by QTOF –MS ES+ (Fig.S34, ESI): m/z, 557.1227,
version 7.0 software.
attributed to [2L1 + Al3++ CH3OH+ H2O]3+ indicating 2:1
stoichiometry (mole ratio) between L1 and Al3+. FTIR spectrum
(Fig.S35, ESI); υ(O-H phenol) 3421, υ(N─H 2º amine) 3251,
Synthesis of L1
The probe, L1 has been synthesised by refluxing the equimolar υ(CH=N) 1600, υ(C─O phenyl) 1303. Thermogram (Fig.S36, ESI)
mixture of salisaldehyde and N-phenyl ethylenediamine in ethanol indicates 3.776% weight loss in the temperature range 30.0-60.3ºC,
for 6h (Scheme 1). The brown-yellow solid is obtained after attributed to the loss of non-coordinated water molecule. Moreover,
evaporation of the solvent. This product is dissolved in methanol and 5.527% weight loss in the temperature range, 60.3-90.5ºC
kept at room temperature for slow evaporation. After several days, corresponds to the loss of two OH groups and 42.992% weight loss
rectangular yellow crystals suitable for single crystal X-ray in the temperature range 90.5-277.9ºC is due to the loss of one L1
diffraction are collected. Yield is 95%. Anal. calcd (%): C, 74.97; H, unit.14.044% weight loss in the temperature range, 277.9-476.6ºC
6.71 and N, 11.66; found: C, 75.02; H, 6.67 and N, 11.80. The X-ray corresponds to the loss of aldehyde unit as a result of rupture of
structure is presented in Fig.S23 (ESI). Significant crystal and CH=N bond of the remaining L1 moiety. 22.771% weight loss in the
1
refinement parameters are summarized in Table S3 (ESI). H NMR temperature range, 476.6-572.1ºC is due to the loss of aldehyde part
(Fig.S24, ESI) (300 MHz, CDCl3), δ (ppm): 3.49 (2H, t, J = 6.0 Hz), through rupture of CH=N bond of the remaining organic amine part.
3.79 (2H, t, J = 6.0 Hz), 6.62 (1H, d, J = 0.6 Hz), 6.65 (1H, d, J = 0.9 The presence of non-coordinated water molecule in the [L1+Al3+]
Hz), 6.72 (1H, t, J = 6.0 Hz), 6.85 (1H, t, J = 6.0 Hz), 6.95 (1H, d, J complex has also been supported by this thermal analysis. UV-Vis.
= 9.0 Hz), 7.16 (1H, d, J = 1.8 Hz), 7.20 (1H, t, J = 1.5 Hz), 7.24 (Fig.S37, ESI): λ (nm) in EtOH/ H2O, 1/1, v/v (ε, M−1 cm−1): 385 nm
(1H, t, J = 1.8 Hz), 7.29 (1H, t, J = 6.0 Hz), 7.54 (1H, t, J = 1.2 Hz), (32500), 273 nm (132500). Excitation spectrum using emission
8.32 (1H, s), 9.90 (1H, s). 13C NMR (Fig.S25, ESI) (300 MHz, wavelength is presented in Fig.S37 (ESI).
CDCl3), QTOF – MS ES+ (Fig.S26, ESI): [M + H]+ = 241.6. FTIR The [L1+Al3+] adduct is also prepared by stirring equivalent amount
(cm−1) (Fig.S27, ESI): υ(O-H phenol) 3383, υ(N─H 2º amine) 3360, of Al(NO3)3.9H2O with the methanol solution of [L1+Zn2+] complex
υ(CH=N) 1629, υ(C─O phenyl) 1315. Thermogram (Fig.S28, ESI) at room temperature. Evaporation of the solvent generates a brown
indicates the stability of L1 up to 200ºC. UV-Vis. (Fig.S29, ESI): λ solid having QTOF –MS ES+ (Fig.S38, ESI) at m/z, 557.1225,
(nm) in EtOH (ε, M−1 cm−1), 404 nm (8500), 305 nm (32500), 250 conforming the formation of [L1+Al3+] adduct.
nm (132500). Excitation spectrum using emission wavelength is
presented in Fig.S29 (ESI).
Cell imaging studies
Human breast cancer cell line MCF7 are grown in DMEM
(Sigma, St. Louis, USA) supplemented with 10% fetal bovine serum
Synthesis of [L1+Zn2+] complex
Methanol solution of Zn(NO3)2.6H2O (50 mg, 0.1680mmol) is (Sigma, St. Louis, USA), 2 mM glutamine, 100 U/ mL penicillin-
added drop-wise to a magnetically stirred solution of L1 (80 mg, streptomycin solution (Gibco, Invitrogen, USA) in presence of 5%
0.3361 mmol) in methanol at room temperature. After 2-3 min, a CO2 at 37°C. For in vitro imaging studies, the cells are seeded in 6
white precipitate that appeared is filtered and dissolved in minimum well culture plate with a seeding density of 105 cells per well. After
volume methylene chloride and kept at room temperature for few reaching 60%–70% confluence, the previous media is replaced with
days to obtain rectangular white crystals, suitable for X-ray serum free media, supplemented Zn2+, Al3+ and L1 at a concentration
crystallography. Yield is 80%. Anal. calcd (%): C, 66.24; H, 5.56 of 50, 50 and 20 µM, and incubated for 2h to facilitate their uptake
and N, 10.30; found: C, 66.12; H, 5.49 and N, 10.43. Significant by cells. The cells are then observed under an inverted microscope at
crystal and refinement parameters are summarized in Table S3 (ESI). different magnifications to examine any adverse effect on cellular
The QTOF–MS ES+ (Fig.S30, ESI) at m/z, 543.30 is assigned to morphology. L1 treated cells are then incubated with Zn2+ and Al3+
[2L12─ + Zn2+ + H+]+, confirming 2:1 (mole ratio) stoichiometry alone for 15 – 30 min or with Zn2+ followed by Al3+and observed
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under fluorescence microscope. Control experiment is performed 11 H. Sharma, N. Singh and D.O. Jang, Tetrahedron Letters, 2014,
using the said medium devoid of any metal salts.
55(49), 6623.
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DOI: 10.1039/C5DT01303H
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Fig.11 Fluorescence image of the MCF7 cells after incubation for 2h with (a)
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Conclusions
In summary, L1 is an efficient ratiometric sensor for Zn2+ that can
detect Al3+ as well and function through ESIPT and CHEF
mechanism. Moreover, [L1-Zn2+] complex has higher sensitivity for
Al3+ functioning via displacement approach. Both the structures for
L1 and [L1-Zn2+] complex have been authenticated by single crystal
X-ray analysis. The sensing of Zn2+ and Al3+ has been digitalized
through construction of a binary logic gate (combination of OR and
NOT gates) that function as a molecular switch. The excellent
sensing process has been materialized in fluorescence cell imaging
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(UGC) for infrastructural facilities. We are grateful to Dr.
Arnab Banerjee, University of Texas, Austin for cell imaging
studies. We sincerely acknowledge Dr. S. Karmakar, USIC, BU
for help regarding XRD analysis.
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