Rhodamine-azobenzene based single molecular probe for multiple ions sensing: Cu2+, Al3+, Cr3+ and its imaging in human lymphocyte cells

Article abstract of DOI:10.1016/j.saa.2019.04.056

A photoinduced electron transfer (PET) and chelation-enhanced fluorescence (CHEF) regulated rhodamine-azobenzene chemosensor (L) was synthesized for chemoselective detection of Al³⁺, Cr³⁺, and Cu²⁺ by UV–Visible absorption study whereas Al³⁺ and Cr³⁺ by fluorimetric study in EtOH-H₂O solvent. L showed a clear fluorescence emission enhancement of 21 and 16 fold upon addition of Al³⁺ and Cr³⁺ due to the 1:1 host-guest complexation, respectively. This is first report on rhodamine-azobenzene based Cr³⁺ chemosensor. The complex formation, restricted imine isomerization, inhibition of PET (photo-induced electron transfer) process with the concomitant opening of the spirolactam ring induced a turn-on fluorescence response. The higher binding constants 6.7 × 10³ M^?1 and 3.8 × 10³ M^?1 for Al³⁺ and Cr³⁺, respectively and lower detection limits 1 × 10^?6 M and 2 × 10^?6 M for Al³⁺ and Cr³⁺, respectively in a buffered solution with high reversible nature describes the potential of L as an effective tool for detecting Al³⁺ and Cr³⁺ in a biological system with higher intracellular resolution. Finally, L was used to map the intracellular concentration of Al³⁺ and Cr³⁺ in human lymphocyte cells (HLCs) at physiological pH very effectively. Altogether, our findings will pave the way for designing new chemosensors for multiple analytes and those chemosensors will be effective for cell imaging study.

Full text of DOI:10.1016/j.saa.2019.04.056

Accepted Manuscript
Rhodamine-azobenzene based single molecular probe for multiple
ions sensing: Cu2+, Al3+, Cr3+ and its imaging in human
lymphocyte cells
Subhabrata Mabhai, Malay Dolai, Surya Kanta Dey, Anamika
Dhara, Sujata Maiti Choudhury, Bhriguram Das, Satyajit Dey,
Atanu Jana
PII:
DOI:
Reference:
S1386-1425(19)30437-8
https://doi.org/10.1016/j.saa.2019.04.056
SAA 17064
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 4 March 2019
Revised date:
Accepted
date:
18 April 2019
19 April 2019
Please cite this article as: S. Mabhai, M. Dolai, S.K. Dey, et al., Rhodamine-azobenzene
based single molecular probe for multiple ions sensing: Cu2+, Al3+, Cr3+ and its imaging
in human lymphocyte cells, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, https://doi.org/10.1016/j.saa.2019.04.056
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ACCEPTED MANUSCRIPT
Rhodamine-azobenzene based single molecular probe for multiple ions
sensing: Cu2+, Al3+, Cr3+ and its imaging in human lymphocyte cells
Subhabrata Mabhai,a Malay Dolai,b Surya Kanta Dey,c Anamika Dhara,d Sujata Maiti
Choudhury, c Bhriguram Das,e Satyajit Dey,*,e Atanu Jana*,f
aDepartment of Chemistry, Mahishadal Raj College, East Midnapore, Mahishadal, West
Bengal, Pin No. 721628, India.
bDepartment of Chemistry, Prabhat Kumar College, Contai, Purba Medinipur 721401, India.
cDepartment of Human Physiology with Community Health; Vidyasagar University,
Rangamati, Medinipur, West Bengal, Pin No. 721102, India.
dDepartment of Chemistry, Jadavpur University, Raja S. C. Mallick Road, Kolkata 700032,
India.
eDepartment of Chemistry, Tamralipta Mahavidyalaya, East Midnapore, West Bengal, Pin
No. 721636, India. E-mail: satyajitdeyoc@gmail.com
fCenter for Superfunctional Materials, Department of Chemistry, School of Natural Science,
Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, South Korea
E-mail: atanujanaic@gmail.com
Abstract
A photoinduced electron transfer (PET) and chelation enhanced fluorescence (CHEF)
regulated rhodamine-azobenzene chemosensor (L) was synthesized for chemoselective
detection of Al3+, Cr3+, and Cu2+ by UV-Visible absorption study whereas Al3+ and Cr3+ by
fluorimetric study in EtOH-H2O solvent. L showed a clear fluorescence emission
enhancement of 21 and 16 fold upon addition of Al3+ and Cr3+ due to the 1:1 host-guest
complexation, respectively. This is first report on rhodamine-azobenzene based Cr3+
chemosensor. The complex formation, restricted imine isomerization, inhibition of PET
(photo-induced electron transfer) process with the concomitant opening of the spirolactam
ring induced a turn-on fluorescence response. The higher binding constants 6.7 × 103 M-1 and
3.8× 103 M-1 for Al3+ and Cr3+, respectively and lower detection limits 1 ×10-6 M and 2 ×10-6
M for Al3+ and Cr3+, respectively in a buffered solution with high reversible nature describes
the potential of L as an effective tool for detecting Al3+ and Cr3+ in a biological system with
higher intracellular resolution. Finally, L was used to map the intracellular concentration of
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Al3+ and Cr3+ in human lymphocyte cells (HLCs) at physiological pH very effectively.
Altogether, our findings will pave the way for designing new chemosensors for multiple
analytes and those chemosensors will be effective for cell imaging study.
1. Introduction
Environmental pollution caused by the high concentration of heavy metals in sewage remains
a global challenging problem. Heavy metal ions like Cu2+, Al3+, and Cr3+ provide essential
indispensable nutrients for life when taken in regulated amount, but their uncontrolled or
overexposure generates acute biological and physiological disorder [1–3]. Cu2+, a cofactor of
various enzymes like cytochrome co-oxidase, tyrosinase, and superoxide dismutase, plays a
significant role in a physiological system such as iron regulation [4–6]. It turns as a toxic
element when taken in excess of recommended amounts of 1.3 ppm (~20 μM) in drinking
water and generated oxidative stress neuro degenerative diseases such as Alzheimer’s
disease, Wilson’s disease, Menke’s disease, gastro intestinal lipid metabolism disorders [7].
The toxic carcinogenic Cr6+ is converted to Cr3+ by bacterial reduction and bind
nonspecifically with DNA to damage the intracellular activity like DNA replication and
transcription [8]. Although Cr3+ is physiologically less harmful as compared to Cr4+ and Cr6+,
its deficiency increases the risk of diabetes, cardiovascular disease, and cancer [9]. Extensive
use of other metal ion such as aluminum in modern life increases the risk of aluminum
toxicity which bring calcium metabolism disorder, interferes the concentration of iron in the
blood causing Osteomalacia, microcytic hypochromic anemia. Unregulated intake of
aluminum cause encephalopathy, myopathy, dementia, Guamanian amyotrophic lateral
sclerosis, Parkinson’s disease, and Alzheimer’s disease [10]. Therefore, a new challenging
field is emerged for developing artificial chemosensor capable of recognizing the
environmentally and physiologically important analytes in a rapid, inexpensive and sensitive
way [11]. Due to high selectivity, sensitivity and easy operational use over other methods,
colorimetric and fluorometric probes become the first choice for practical applications [12].
Meanwhile, the idea of designing a new cost-effective molecular probe which binds more
than one analyte using a single detection method or an array of detection methods gain the
importance over one-to-one normal sensors [13–15]. To date, in comparison to diamagnetic
Al3+, there are very few ‘Switch-On’ sensors reported for paramagnetic Cu2+ (d9) and Cr3+(d3)
because of their fluorescence quenching property [16]. Higher photostability and longer
absorption-emission wavelength along with the ability to trigger a dramatic change in colour
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by the opening of the non-fluoroscent closed shell to zwitterionic fluorescent spirolactam
form make Rhodamine derivatives very reliable platform for constructing Off-On
fluorescence sensor [17,18].
In this work, we have designed and synthesized a new (E)-2-hydroxy-3-methoxy-4-
((4-nitrophenyl)diazenyl)benzaldehyde-appended rhodamine hydrazone derivative (L) in a
mixed solvent as a single chemosensor for sensing multiple analytes such as Cu2+, Al3+ and
Cr3+. The outline of the synthesis is depicted in Scheme 1. The sensor L exhibits the ‘Turn-
On’ property via the chelation-enhanced fluorescence (CHEF) process [19] and inhibited
PET which was supported by the different photophysical process. We have constructed
INHIBIT molecular Logic gate using the sensing properties. The experimental findings are
well-correlated with theoretical results using density functional theory calculations. In addition, L
was successfully applied in the imaging of Al3+ and Cr3+ in human lymphocyte cells (HLCs).
2. Experimental Section
2.1 Materials and instruments
3-Ethoxysalicylaldehyde (99 %), p-nitro aniline (97 %), Rhodamine B (99.0 %) from Sigma-
Aldrich, high-purity HEPES (99.0%), Na2EDTA(98.0%), perchlorates and nitrate salts of the
different metal cations (Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Cr3+, Al3+, Mn2+, Fe3+, Zn2+, Ni2+, Pd2+,
Mg2+, Pb2+ Na+, Hg2+) and all solvents were purchased from Merck (India) Ltd and were used
,
as received without further purifications. All spectroscopic data were recorded using HPLC
grade solvent. UV-Vis absorption study was performed with Evolution-201 spectrometer and
XENO Flash (PTI) fluorescence spectrophotometer with quartz cuvette (path length = 1 cm)
was utilized to record the emission spectra with the excitation wavelength 520 nm for both
ligand and complex at room temperature.1H NMR spectra and 13C NMR spectra were
recorded on Brucker 400 MHz instruments using TMS as an internal standard. Chemical
1
shifts (δ) were reported in ppm units and H–1H coupling constants in Hz. IR spectra (KBr
pellet, 400–4000 cm–1) were recorded on a Perkin-Elmer infrared spectrophotometer (Model:
883). Elemental analysis for carbon, hydrogen, and nitrogen was performed with Perkin
Elmer 2400 CHN analyzer. Electrospray ionization MS (ESI-MS) on Qtof Micro YA263
mass spectrometer. Fluorescence Quantum Yield was calculated using Fluorescein an
optically matching standard (Φr = 0.79). HORIBA Jobin Yvon Fluorocube-01-NL
fluorescence lifetime spectrometer was used to record the time Correlated Single Photon
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Counting (TCSPC) data. Gaussian software was used to have DFT data. Cell imaging was
carried out with Fluorescence microscope (LEICA DFC295, Germany).
2.2 Isolation and culture of human lymphocyte cell (HLCs)
5 ml of blood samples were collected from healthy young volunteers according to the method
of Hudson and Hay [20] about 4 ml of blood were layered onto the same amount of
histopaque 1077 (Sigma-Aldrich Co. LLC, US) and centrifuged at 2000 rpm for 30 min at
room temperature. To a centrifuge tube, the upper lymphocytes monolayer was transferred
and washed three times in phosphate buffer solution (pH 7.4). The human lymphocyte cells
(HLCs) were cultured by resuspending in RPMI medium supplemented with 10 % FBS and
incubated for 24 h at 37 °C in a 95 % humidified and 5 % CO2 atmosphere in a CO2
incubator and used for the experimentation.
2.3 Cell cytotoxicity study
In order to measure the cytotoxicity of the ligand, yellow colored tetrazolium salt 3-(4, 5-
dimethylthiazol-2-yl)-2S-diphenyltetrazolium bromide (MTT) assay was executed in human
lymphocyte cells (HLCs) according to standard procedure [21]. In brief, overnight culture of
HLCs were seeded in 96-well plates at a density of 1×106 cells per well in 100 μL of culture
medium and incubated with a series of concentrations of L (5, 10, 20, 50 and 100 µM) at 37
°C in a 5 % CO2 atmosphere for 24 h. After the removal of the culture medium, cells were
washed by PBS (pH 7.4). 10 µl of MTT solution (1mg mL-1) in PBS was added in each well
and cell were again incubated for 3h at 37 °C. After the incubation, 0.1 % DMSO was added
to each well. The absorbance of the intracellular formazan crystal (blue-violet) was measured
at the wavelength of 540 nm by ELISA ANALYSER (Bio-Rad, Model 680). The cell
viability was quantified as the optical density ratio of the ligand treated group to HLC-
control. Values are mean (M) ± standard error of mean (SEM) of three independent
experiments.
% cell viabilty = (OD of the sample) / (OD of control)×100
The cell cytotoxicity was calculated using the following formula:
% cell cytotoxicity = 100 %- % cell viability
2.4 Computational method
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DFT study is an important tool to obtain better insights into the geometry, electronic
structure, and optical properties of these systems. Ground state electronic structure
calculations in gas phase of both the complexes have been carried out using DFT [22] method
associated with the conductor-like polarizable continuum model (CPCM) [23–25]. Becke’s
hybrid function [26] with the Lee-Yang-Parr (LYP) correlation function [27] was used
through the study. The geometry of the ligand and complex was fully optimized without any
symmetry constraints. On the basis of the optimized ground state geometry, the absorption
spectral properties in ethanol (EtOH) media were calculated by time-dependent density
functional theory (TDDFT) [28–30] approach associated with the conductor-like polarizable
continuum model (CPCM). We computed the lowest 40 singlet – singlet transition and results
of the TD calculations were qualitatively very similar. The TDDFT approach had been
demonstrated to be reliable for calculating the spectral properties of many transition metal
complexes [31–33]. Due to the presence of electronic correlation in the TDDFT (B3LYP)
method, it yields more accurate electronic excitation energies. Hence TDDFT had been
shown to provide a reasonable spectral feature for our complex of the investigation.
We have run the Gaussian for geometry optimization of both the complexes in ground state
with 6-31G under B3LYP. All the calculations were performed with the Gaussian 09W
software package [34]. GaussSum 2.1 program [35] was used to calculate the molecular
orbital contributions from groups or atoms.
2.5 Fluorescence lifetime measurements
Fluorescence lifetimes were measured by the method of Time-Correlated Single-
Photon Counting (TCSPC) using a HORIBA JobinYvon Fluorocube-01-NL fluorescence
lifetime spectrometer. The sample was excited using a laser diode at 450 nm and the signals
were collected at the magic angle of 54.7 ° to eliminate any considerable contribution from
fluorescence anisotropy decay [36]. The typical time resolution of our experimental setup is ~
100 ps. The decays were deconvoluted using DAS-6 decay analysis software. The
acceptability of the fits was judged by χ 2 criteria and visual inspection of the residuals of the
fitted function to the data. Mean (average) fluorescence lifetimes were calculated using the
following equation [37,38].
  2
 


i
i
av 
i
i
in which αi is the pre-exponential factor corresponding to the ith decay time constant, τi.
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2.6 Synthesis of Rhodamine B hydrazide:
Rhodamine B hydrazide was synthesized according to previously reported method
[39].
2.7 Synthesis of 3-ethoxy-2-hydroxy-4-((4-nitrophenyl) diazenyl) benzaldehyde
The diazo compound was synthesized according to the literature procedure [40]. A clear
green solution of p-nitroaniline (0.414 g, 3 mmol) in HCl (30 %, 6 ml) was prepared in
between 0-5 °C by keeping the beaker on ice-salt bath with constant stirring. Colour of the
solution changed from green to pale yellow during dropwise addition of cold aqueous NaNO2
(0.207 g, 3 mmol). This diazotized solution was added to the alkaline solution of 3-ethoxy
salicylaldehyde (0.5 g, 3 mmol) in water (20 ml) containing NaOH (0.12 g, 3 mmol) and
(0.17 g, 1.63 mmol) at 5 °C. After overnight stirring of the resulting solution at room
temperature a dark brown cake was produced, and it was filtered using Whatman-41. The
dark-brown residue was reslurried after repeating washing by 10 % NaCl. The solution made
slightly acidic (pH 4-5) by adding 2-3 drops of conc. HCl. Thereafter the yellow colour dye
was dried by keeping in a vacuum desiccator and purity was confirmed by chromatography.
The yield of the product was 85 %. 1H NMR: [Fig. S1, ESI] [400 MHz, DMSO-d6, δ (ppm)]
δ10.32 (1H, s, Ar-CHO), 8.35-8.33 (d, 2H, J = 8 Hz, Ar-H) 7.98-7.95 (d, 2H, J = 12 Hz, Ar-
H) 7.87 (s, 1H, Ar-H) 7.62 (s, 1H, Ar-H) 4.18-4.13 (8H, q, J = 12Hz,-OCH2) 1.38-1.35
(3H,t, J = 8Hz,-OCH2CH3). Melting point (M.P.) 184 °C.
2.8 Synthesis of chemosensor L
An easy and convenient one-step condensation method was applied to the synthesis of L from
Rhodamine B hydrazide and aldehyde precursor (Scheme 1). Rhodamine B hydrazide (0.460
g, 1.008 mmol) and 2-ethoxy-6-methyl-4-((4-nitrophenyl)diazenyl)phenol (0.319 g, 1.008
mmol) were dissolved in dry EtOH (20 ml) by stirring at room temp. The resulting solution
was refluxed for 6-hrs with constant monitoring by thin layer chromatography (TLC). At the
end of the reaction, the volume of the reaction mixture is reduced to 1/3 of its original volume
so that a deep red colored solid appeared. The deep red colored solid was separated by simple
filtration and washed repeatedly by cold EtOH-Ether (1:1 ratio) after cooling the reaction at
room temperature. Yield ~ 85 %. M.P. 220 °C
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Scheme 1. Synthesis of chemosensor L
1H NMR [Fig. S2, ESI] [400 MHz, DMSO-d6, δ (ppm)] δ10.92 (1H, s, -OH), 9.16 (1H, s, –
CH=N), 8.39-8.37 (2H, d, J = 8 Hz), 8.00-7.98 (2H, d, J = 8Hz), 7.94-7.92 (1H, d, J = 8 Hz),
7.76 (1H, s), 7.63-7.56 (2H, m), 7.43 (1H, s), 7.11-7.10 (1H, d, J=4Hz), 6.46-6.44 (4H, m),
6.37-6.34 (2H, dd, J = 12 & 8Hz), 4.11-4.10 (2H,q, J = 8 Hz), 2.50-2.48 (8H, q, J = 4Hz,-
OCH2), 1.35-1.31 (3H,t, J = 8Hz,-OCH2CH3), 1.07-1.03 (12H, t, J = 8Hz,-NCH2CH3).13C
NMR [Fig. S3, ESI] [125 MHz CDCl3, δ (ppm)] :12.90, 19.53,14.92, 44.15, 64.81, 66.03,
97.90, 105.23,108.70,122.88,123.65, 124.13, 124.36, 125.40,125.53, 127.66, 128.11, 128.41,
133.88, 134.65, 144.73,145.31,146.08, 149.07,152.04,153.14,159.70,164.32. Elemental
analysis (calcd. %) for C43H43N7O6: C, 68.51; H, 5.75; N, 13.01; O, 12.73; found (%): C,
68.48; H, 5.78; N, 13.03; O, 12.70. ESI-MS: [Fig. S4, ESI] m/z calculated for C43H43N7O6
[M+H]+ :754.33, found 754.66.
2.9 Synthesis of L-Al3+ and L-Cr3+Complex
Al(NO3)3.9H2O (0.19 g, 0.506 mmol) and CrCl3 (0.080 g, 0.506 mmol) were added to the two
separate R.B. each containing (0.381 g, 0.506 mmol) of L in acetonitrile. The solution was
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filtered after overnight stirring at room temperature. The deep maroon colored solid filtrate
appeared in both case after several time washing with MeOH kept in vacuum for drying.1H
NMR [Fig. S5, ESI] [400 MHz, DMSO-d6, δ (ppm)] δ 9.44 (1H, s, –CH=N), 8.41-8.39 (2H,
d, J = 8 Hz), 8.00-7.93 (3H, M), 7.78 (1H, s), 7.62-7.60 (2H, m), 7.44 (1H, s), 7.12-7.10 (1H,
d, J = 8 Hz), 6.46-6.44 (4H, m), 6.37-6.32 (2H, m,), 4.11-4.09 (2H,q, J = 4 Hz), 2.51-2.49
(8H, q, J = 4Hz,-OCH2), 1.36-1.33 (3H,t, J = 8Hz,-OCH2CH3), 1.08-1.04 (12H, t, J = 8Hz,-
NCH2CH3). 13C NMR [Fig. S6, ESI] [125 MHz CDCl3, δ (ppm)] : 12.86, 14.06, 14.86,
44.15, 64.82, 97.90, 108.74, 120.94, 120.91, 122.24, 123.64, 124.02, 125.51, 128.10, 128.71,
129.44, 129.97, 132.67, 134.68, 144.72, 145.22, 148.45, 148.57, 151.95, 153.11, 157.27,
157.87, 158.28, 162.07.13C NMR [Fig. S7, ESI] [125 MHz CDCl3, δ (ppm)] :12.94, 15.31,
39.90, 40.06, 40.22, 40.39, 40.55, 40.72, 40.88, 44.20, 64.83, 97.87, 104.70, 105.13, 108.70,
120.06, 120.78, 1213.74, 124.34, 125.60, 128.09, 128.69, 131.46, 145.22, 145.25, 14.17,
148.41, 148.56, 149.06, 151.93, 153.11, 164.40.ESI-MS: [Fig.S8, S9, ESI] m/z calculated for
C43H43N7O6 [L+Al+NO3+H+]+ : 841.1625, found 841.1625 and for C43H43N7O6 [L+Cr+] :
804.51 found 804.51.
2.10 Association constant
The association constant value (Ka) was determined from absorption data using the following
Benesi–Hildebrand (B–H) equations [41].
1
1
1


(A A ) [K(Amax  A )C] (Amax  A )
0
0
0
where A0 is the absorbance maxima of sensor L, A is the observed absorbance at that
particular wavelength at different concentration of the metal ion [C], Amax is the maximum
absorbance value at λmax = 582 nm and 581 nm for Al3+ and Cr3+, respectively during titration
with varying [C], K is the association constant and was determined from the ratio of slope
and intercept of the linear plot, and [C] is the concentration of the Mn+ ion added during
titration studies.
The binding constant is determined from fluorescence intensity data using Benesi–
Hildebrand equation:
1
1
1


(I  I0 ) (I1  I0 ) (I1  I0 )k[M n ]m
where [Mn+] is the metal ion concentration, I0, I and I1 indicate emission intensities in the
absence of, at intermediate and at infinite concentrations of metal ions, respectively. An
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aqueous stock solution of Mn+ in H2O with an exact concentration of 1 × 10−3 mol L−1, in an
aqueous HEPES buffer (pH 7.2) and an effective concentration of 2 × 10−5 mol L−1 ligand
solution was prepared in the EtOH-H2O medium at pH 7.2 for this purpose.
A linear relationship between 1/(A-A0) vs. 1/[Mn+] plot indicates 1:1 binding stoichiometry
and the binding constant calculated to be 6.7435 × 103 M-1, 3.8× 103 M-1 respectively for Al3+
and Cr3+ and 5.685×103 M-1 for Cu2+ (Fig. S10, S11, S12 ESI).
2.11 Procedure for metal ion sensing
A stock solution of the sensor L was prepared with concentration 1×10-3 in Ethanol-aqueous
HEPES buffer (5µM, pH 7.2, 9:1, v/v) for both spectroscopic studies. Metal solution of
different conc. (C = 1×10-3, C = 1×10-2) were prepared from Ethanol-de-ionized water.
Absorbance and fluorometric selectivity were examined by taking 20 µL of the stock solution
in 2 ml ethanol in a quartz cuvette (path length, 1 cm) maintaining the final conc.10×10-6 M
along with 50µL (C = 1×10-2) metal solution. Titration experiment was carried out by the
gradual addition of metal ion (C = 1×10-3) to sensor solution.
2.12 Fluorescence Quantum Yield
Fluorescein an optically matching standard (Φr = 0.79) was used in spectroscopic grade
EtOH to determine the Fluorescence Quantum Yield by the following Equation [42].
2
A F s
r
s  r (
s )(
)
2
A F r
s
r
Where As and Ar are the absorbance of the sample and reference solutions, respectively, at
the same excitation wavelength, Fs and Fr are the corresponding relative integrated
fluorescence intensities, and η is the refractive index of the solvents. The Quantum Yield of
ligand Φ = 2.94 %, changes to 11 fold Φ = 32.6 % for Al3+ and 3.61 fold Φ = 10.6 % for Cr3+.
2.13 Binding ratio measurements (Job’s plot)
Job’s plot measurement was made from UV-Vis titration. Receptor solution was taken in 10
vials at regular intervals starting from 20 µL to 2 µL. whereas metal solution was injected
into it in reverse order at the same regular interval. The total solution volume of each vial is
fixed to 2mL. Absorption data was recorded out at room temperature after shaking the vials
for 2 minutes [43].
3. Results and discussion
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3.1 Selectivity studies
Selectivity of L is ascertained spectrometrically by taking 25 equivalent of several
competitive metal ions (Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Cr3+, Al3+, Mn2+, Fe3+, Zn2+, Ni2+, Pd2+,
Mg2+, Pb2+ Na+, Hg2+) in a solution containing 20 µL L in 2 mL EtOH-H2O (9:1,v/v, 5µM
,
HEPES pH 7.2) solvent. The result exhibited that Al3+, Cr3+ and Cu2+ exhibit sharp change in
absorbance whereas Al3+ and Cr3+ show distinct change in PL intensity with respect to other
metal ions. Al3+, Cr3+, and Cu2+ altered the absorbance spectral line with the appearance of
three bands at λmax = 551, 554, and 557 nm, respectively under the identical condition. Under
this optimal condition (i.e.; pH = 7.2) L changes its fluorescence spectral line in presence of
Cr3+and Al3+ at 581 nm & 582 nm, respectively. The superior dual fluorometric selectivity of
L was experienced in the naked eye after 366 nm radiation of UV light where the color of the
L changed immediately from colourless to deep pink after the addition of Al3+ and Cr3+. Such
interesting findings motivate our groups to explore the L as a single probe for dual metal
sensing by fluorometric method. Though in ambient light, L shows colorimetric distinction
with Ba2+, Ca2+, Co2+ (dark yellow), Fe3+and Pd2+ along with the Al3+, Cr3+, and Cu2+ metal
ions (Fig. 1), but in UV-Visible absorption study, our sensor shows a prominent change in
absorbance only with three metal ions (Cu2+, Al3+, and Cr3+) whereas Cr3+and Al3+ are
distinguished from other metal ions by PL study. Oxophilic, diamagnetic, hard acid Al3+ with
its high ionic potential and low ionic radius provides strong binding energy with the hard
base than other paramagnetic divalent and trivalent metal ions, and hence make them as a
strong guest for L [43,44]. Again particularly intersystem crossing in excited singlet state by
transferring electron from paramagnetic Cu2+ (d9) to the Rhodamine-B fluorophore promotes
the intersystem absorption phenomena making it a natural fluorescence quencher [45],[46].
3.2 Spectroscopic recognition.
3.2.1 UV-Visible absorption studies:
The extraordinary dual sensitivity by this newly designed rhodamine-based probe was
investigated by UV-Vis and fluorescence method, using different metal solution in EtOH-
H2O (9:1 V/V) at optimized pH 7.2. The color of L changes upon addition of Ba2+, Ca2+,
Co2+, Fe3+, and Pd2+ along with the Al3+, Cr3+, and Cu2+ metal ions but only Cr3+ and Al3+
exhibit pink color under UV light (366 nm) (Fig. 1). This optical discrimination was further
justified during the quantitative recording of UV-Visible absorption spectra of several
analytes viz. Na+, K+, Ba2+, Ca2+, Cd2+, Co3+, Cr3+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+,
Pd2+, Zn2+ and Al3+ in aforementioned ligand solution taken in a quartz tube containing 2 ml
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EtOH-H2O (9:1, v/v) pH = 7.2 using 5 µM HEPES buffer at 25 °C. Interestingly, as depicted
in Fig. 2, UV-Visible spectral results revealed that among the all coexisting metal ions only
Cu2+, Al3+, and Cr3+ alters the spectral line at λmax = 551, 554 and 557 nm, respectively. To
have a better quantitative appraisal of the reversible interaction of the sensor L, absorption
spectral variation was carried out between sensor L with Mn+and resulting chelated complex
with a more powerful hexadentate chelating agent Na2EDTA. Gradual incremental addition
of Al3+, Cr3+, and Cu2+, to the fixed solution of sensor L (2 ml) in EtOH-H2O (9:1, v/v) at pH
= 7.2 a progressive increase of the absorption peak for the guest metals were observed along
with deepening of pink color. After addition of 46 μM Al3+, 36 μM Cr3+ and 50 μM Cu2+, no
further increment was observed at λmax = 554, 557 and 551 nm, respectively, for Al3+, Cr3+
and Cu2+.
Fig. 1. Visual display of L plus metal ions: (a) under normal illumination (b) and
demonstrating fluorescence under 365 nm illumination.
This indicates the chelating saturation of sensor L for guest metal ions due to the
transformation of Rhodamine B from close spirolactam ring to stable metal-induced open
conjugated xanthene form (Fig. 3 (a-c)) [47]. However, complete bleaching of pink colour, as
11

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well as the disappearance of aforementioned peaks (Fig. 3 (d-f)) by the incremental addition
of Na2EDTA, clearly indicates reversible binding nature of sensor ligand towards the guest
cation. Association constant was calculated from absorbance vs. [Mn+] curve by Benesi-
Hildebrand linear curve fitting methods and was found to be ka = 6.7435×103 M-1, 3.8×103M-
1, and 5.685×103 M-1, respectively, for Al3+, Cr3+, and Cu2+. (Fig. S10, S11, S12 ESI).The
calculated LOD value 1.07x10-6 M, 2.02 x10-6 M, 1.6×10-6 M for Al3+, Cr3+ and Cu2+ based on
UV-Visible absorption data by 3σ method [48] also satisfies the limit of drinking water for
both Al3+ and Cr3+ (Fig. S13, S14, S15 ESI).
As our purpose is to establish the designed sensor as an effective fluorescent probe so we are
not interested to explore the fluorometric sensing property of Cu2+ as it does not obliterate the
fluorescent spectral nature of the sensor L.The selectivity tuning of L towards Al3+ and Cr3+
was explored by the fluorometric method.
0.5
2+
Lig
Cu
+
Na
+
K
0.4
2+
Ba
2+
Ca
3+
2+
Cd
Al
3+
CO
0.3
3+
Cr
3+
2+
Cr
Cu
3+
Fe
2+
Hg
0.2
2+
Mg
2+
Mn
2+
Ni
2+
0.1
Pb
2+
Pd
2+
Zn
3+
Al
0.0
450
500
600
Wavelengt5h50(nm)
Fig. 2. Absorption spectral nature of Probe L upon addition of various metal ions (Na+, K+,
Ba2+, Ca2+, Cd2+, Co3+, Cr3+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+, Pd2+, Zn2+, and Al3+
) 25 equiv. in EtOH-H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C
12

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(c)
(b)
0.8
0.2
(a)
0.4
3+
36 Cr
3+
46 Al
0.6
0.4
0.2
0.0
50M Cu2+
0.3
0.2
0.1
0.0
0.1
0.0
3+
3+
0 Cr
0  Al
0M
400
450
550
600
wavel5e0n0gth (nm)
450
500
550
600
Wavele5n5g0th (nm)
500
600
Wavelength(nm)
(d)
(f)
(e)
0.4
0.8
0.2
0.1
0.0
0 EDTA
0 EDTA
0M
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
46EDTA
38 EDTA
20M
500
600
Wavele5n5g0th (nm)
450
500
550
600
500Wavelength (nm)
550
600
Wavelength(nm)
Fig. 3. UV-Vis absorption spectra of sensor Probe L (10µM) upon titration with (a) Al3+ (b)
Cr3+ (c) Cu2+ (c =1.0×10-3 M) in EtOH-H2O [9:1, v/v, 5µM HEPES pH 7.2]. Absorbance
spectra of sensor (d) L-Al3+, (e) L-Cr3+,and (f) L-Cu2+ complex upon incremental addition of
EDTA
3.3 Fluorescence study
A better understanding of emissive behavior of sensor L is explored by choosing an
appropriate solvent. The emissive behavior of L-Al3+ and L-Cr3+in different solvents (EtOH,
MeOH, CH3CN, DMF, and DMSO) were tested with the findings that in both cases EtOH
was the suitable solvent for the study of emissive property (Fig. 4). The selectivity and
sensitivity trait of L towards Al3+ and Cr3+ with respect to other alkali and alkaline earth
metal analytes such as Na+, K+, Ba2+, Ca2+, Cd2+, Co3+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+,
Pb2+, Pd2+ and Zn2+ at pH = 7.2 using 5 µM HEPES buffer at 25 °C was also pursued by
exciting the sensor solution above λmax = 520 nm in optically matching 2 ml EtOH-H2O (9:1,
v/v) solution. Under the experimental condition, no noticeable emission peak for ligand was
observed in the presence of alkali and alkaline earth metal ions except for a very weak
absorption for Cu2+ was detected at λmax = 574 nm and fascinatingly, enough under the same
experimental condition, Al3+ and Cr3+ generate a remarkable fluorescence enhancement of 21
13

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fold and 16 fold at λmax = 582 nm and 581 nm, respectively (Fig. 5) as a result of metal-
induced CHEF process and inhibition of PET process, when 25 eq. of different metal ions
were added to the sensor solution.
150
(c)
100
50
0
THF
DMSO
Ligand
DMF
Al 3+
MeCN
Cr 3+
EtOH
MeOH
Fig. 4. Solvent effect on the emissive behavior of sensor L towards (a) Al3+and (b) Cr3+ ions.
Inset: Photographic image of the emissive pattern in the different solvent system under UV-
cabinet (λ = 366 nm) (a) L-Al3+ (b) L-Cr3+
The onset of Photo-induced electron transfer (PET) process by –C=N bond isomerization
along with a donation of an electron from HOMO of Schiff base N and –OH of hydroxyl
aldehyde to fluorophore LUMO is mainly responsible for ‘Turn-Off’ emissive behavior of the
sensor [49]. Again the inhibition of isomerization and appropriate complexation in presence
of specific guest and functional group attached to the fluorophore makes the PET process
invalid and initiates the chelation-enhanced fluorescence process (CHEF) by transferring
HOMO electron of the fluorophore to its LUMO arresting the previous electron transfer [50]
(Scheme 2). This strong ‘Turn-On’ selectivity of sensor L also observed when sensor
solution treated separately with each of 25eq. Al3+ and Cr3+ under long-range UV lamp of
(366 nm). The almost immediate transformation of colourless sensor solution to pink colour
supports the ‘Turn-On’ property of the sensor.
To elucidate the reversible binding mode of sensor L fluorescence titration was performed
with Al3+ and Cr3+. Fluorescence intensity increases maximally up to λmax = 582 nm and 581
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nm with the gradual addition of 46 µM Al3+and 36 µM of Cr3+ (Fig. 6 (a-b)) after which
increment ceases down with a quantum yield (Φ = 11.08) for Al3+ and (Φ = 3.61) for Cr3+,
respectively. The fluorescence intensity decreases gradually almost by the similar manner
with the incremental addition of Na2EDTA (Fig. 6 (c-d)) due to the removal of metal ions
from the L-M3+ complex by the formation of strong chelate with EDTA. The lower quantum
yield and lower emission intensity of Cr3+in compared to Al3+is attributed to the higher
oxophilic nature higher CFSE value and hard acid character of Al3+, which encourages the
higher CHEF effect of the sensor for Al3+.
3+
Al
Lig
2+
2+
2+
Co
Cd
Mn
Mg
Ba
Ca
2+
2+
2+
3+
Fe
2+
Cu
2+
Pd
3+
Cr
+
K
2+
Ni
+
2+
Pb
2+
Zn
Na
Hg
2+
550
600
650
Wavelength (nm)
Fig. 5. Emission spectral nature of Probe L (10 µM) upon addition of various metal ions
(10 equiv.) in EtOH-H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C
Scheme 2. Schematic Presentation Showing the Possible PET-CHEF mechanism for
Al3+ and Cr3+sensing.
15

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Fig. 6. Emission spectral change of the ligand L (10µM) upon incremental addition of
(a) Al3+ and (b) Cr3+ in EtOH-H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C. Emission
spectral change of the (c) L-Al3+and (d) L-Cr3+complex upon incremental addition of
EDTA in EtOH-H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C.
3.4 Interference studies
For effective use of the probe, achieving high selectivity as a host for Al3+ and Cr3+ in
presence of other potentially challenging metal cation is a very important index. In order to
check the cross sensitivity, we do perform the selectivity of our probe for a wide range of
cations along with Al3+ and Cr3+ in the EtOH-H2O medium. Clearly, it is revealed from the
graph that the emission intensity of the probe remains almost unchanged when 5 equivalents
of other metal ions added to the buffer solution of the probe as that of Al3+ and Cr3+ (Fig. 7).
Surprisingly in both case after the addition of 10 equivalents of Cu2+ and Fe3+, there was a
rapid quenching of fluorescent intensity. Fe3+ quenches the intensity because of the stability
of its half-filled (d5) shell [51] and the onset of non-radiative decay caused by the electron
transfer from redox active (d9) Cu2+ to the excited fluorophore of rhodamine moiety is one of
the probable reasons for fluorescence quenching.
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Therefore, the dual metal experiment clearly evidenced that our probe can be used as a dual
target recognizing agent in a single detection method.
5000
4000
3000
2000
1000
0
comparative ions + Cr3+
comparative ions +Al3+
Fig. 7. Relative emission intensity of L after addition of 5 equivalents of competing for
metal ions as that of Al3+/Cr3+to the solution of L in EtOH-H2O [9:1, v/v, 5 µM HEPES pH
7.2] at 25 °C.
3.5 Reversibility
The practical usability of L depends mainly on its alternating enhancing and reviving
mechanism. This reversibility is investigated to monitor the important changes in the
biological system. Hexadentate chelating agent Na2EDTA bleach the ‘signal-on’ absorption
band of both Al3+ and Cr3+ through the process of demetallation from L-Mn+ complex by
regenerating the spirolactam ring. Our reversibility experiment well substantiates this theory.
Addition of Na2EDTA to the L-Mn+ complex solution completely arrests the fluorescent by
returning it to the 520 nm indicating the generation of metal-free ligand. Turned-on property
of L is regained upon addition of either Al3+ or Cr3+. This result is recreated after sequential
several additions of metals and Na2EDTA, which clearly indicates that the receptor reversibly
recognized Al3+ and Cr3+ (Fig. 8).
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Fig. 8. Changes of emission intensity at 582 nm (λex = 520 nm, 10 μM) in EtOH-H2O (9:1,
v/v at pH = 7.2 HEPES buffer) upon repetitive addition of (a) Al3+ (1.0 equiv.) and EDTA
solution. (b) changes of emission intensity at 581 nm (λex = 520 nm, 10μM) in EtOH-H2O
9:1, v/v at pH = 7.2 HEPES buffer) upon repetitive addition of Cr3+ (1.0 equiv.) and EDTA
solution.
3.6 pH study
The L can only be used effectively in a complex biological system if it can withstand in
physiological pH without hydrolysis by the interfering proton. Although we have used
HEPES buffer solution to arrest the hydrolysis of guest metal ion throughout the sensing
process so as to eliminate any type of chance that may be caused by the participation of
proton in incremental fluorescent luminescence, the pH study has been performed over a
long-range of pH (2-12) in EtOH-H2O (9:1 v/v) medium both in the presence and absence of
metal ions [52]. At lower pH, the protonation induced spirolactam ring opening affects the
gradual increment of fluorescence intensity of probe L irrespective of the inclusion of metal
ions. However, in presence of metal ions, the probe L shows excellent fluorescent sensing
ability which remains almost unaltered throughout the physiological range of pH. On another
site, there is no observable emission for the probe L in absence of analytes within this
physiological range as it remains in its spirolactam form in this pH (Fig. 9) [53]. This very
conclusive study clearly negates any type of interference of the proton in physiological pH
and indicates that the luminescence in the physiological pH is only due to L-Mn+ binding
18

ACCEPTED MANUSCRIPT
spirolactam ring opening. So clearly this novel sensor is a biocompatible one for detection of
Al3+ and Cr3+.
160
In absenceof analytes
3+
In presence of Al
140
3+
In presence of Cr
(a)
120
100
(b)
80
60
40
20
(c)
0
2
4
6
8
10
12
pH
Fig. 9. Fluorosenseresponse of (a) L-Al3+ (brown line) at λmax = 582 nm, (b) L- Cr3+ (pink
line) at λmax = 581 nm, and (c) L (red line) as a function of pH in EtOH-H2O 9:1, v/v at pH
= 7.2 HEPES buffer) at 25 °C at the excitation wavelength of 520 nm. The pH was
adjusted using 1 M aqueous solutions of HCl.
3.7 Plausible mechanism
The conspicuous ‘Turn-On’ behaviour of L towards Al3+ and Cr3+ is a combination of the
PET-CHEF process. The Turn Off fluorescent property which is possibly responsible for a
lower quantum yield of L is due to PET process where lone pair from N center of spirolactam
moves to the azobenzene moiety making the spirolactam tautomer colorless [54]. On the
other hand, colourless non-fluorescent to the pink fluorescent makeover of L is an obvious
result of the binding-induced spirolactam ring opening process [55]. The coordinating site
offered by the host to the guest through keto group and the hydroxyl group is confirmed from
the shifting of keto carbonyl IR peak of spirolactam ring at 1710 cm-1 to 1646 cm-1 and
1647cm-1 for L-Al3+ and L-Cr3+, respectively (Fig. S16, S17, and S18, ESI). The lower wave
no. of C=O and broadening of –OH band in IR spectra, which is practically due to the higher
polarisation of these bond after induction with metal strongly supports the complexation of
metal ion (Al3+, and Cr3+) with the ligand [56,57]. Disappearing of 13C peaks at (δ) 66.03ppm
1
(Fig. S6, S7 ESI) for sp3 carbon and also disappearing of H NMR peak at (δ) 10.92 ppm
19

ACCEPTED MANUSCRIPT
(Fig. S5, ESI)for –OH proton along with shifting of –CH=N peak at (δ) 9.44 ppm supports the
formation of L-Al3+ and L-Cr3+ complexation [58]. Again, the L-Mn+ complexation as well as
the better complexing ability of L-Al3+is also be confirmed from the higher τav value of L-
Mn+ than bare L and higher τav value of L-Al3+ than L-Cr3+complex (Table-5). Furthermore,
the 1:1 binding pattern of the L-Mn+ complex was confirmed from UV–Vis absorption by the
Job’s plot for L and Mn+ (Fig. S19, S20, S21 ESI). A thorough inspection of the Job’s plot
exposed that absorption reaches its maxima near about 0.5-mole fraction of Cr3+ and Al3+
indicating ideal binding mode for L-Al3+ and L-Cr3+ complex, respectively. The superiority of
our sensor L has been established by comparing the detection limit with many other reported probes
[Fig. S22 ESI]
3.8 Logic gate interpretation
The quick and subtle response of our probes towards the dual metal inspired us to check its
reversibility by constructing a double input and single output based noncommutative type
logic gate [59,60]. The INHIBIT logic gate is constructed in the maximum emission of Al3+
and Cr3+ at λem = 582 nm and 581 nm with the basic theory of Boolean algebra where 1
represents ‘YES’/‘ON-state’ and 0 pointing ‘NO’/OFF-state. In the absence of both metal and
EDTA, the output is zero indicating off state. In variation 2 when the input is only metal
without EDTA maximum emission intensity is observed for Al3+ and Cr3+ at λem = 582 nm
and 581 nm, respectively, pointing 1 (on) state, in the third row where EDTA is the only
chemical entity to input,output is zero and in the fourth row where both the metals and EDTA
is used as input, output again becomes zero. Therefore, input 1 performs the yes operation
and the input 2 satisfies the criteria of NOT gate [61]. Considering all the results among the
16th TRUTH Table our truth table matches with the truth table of INHIBIT logic gate (Fig.
10).
(a)
(b)
Fig. 10. (a) Truth table (b) circuit diagram for Inhibit Logic Gate
20

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3.9 Geometry optimization and electronic structure
The optimized geometries of the Al3+ complex and its Cr3+ complex are shown in Fig. 11.
The ground state geometry optimization for the complexes 1 and 2 were performed in the gas
phase. Main optimized geometrical parameters of the complex 1 are listed in Table 1 and for
complex 2 in Table 2. In case of complex 1, the Al3+ center is penta-coordinated with one di-
deprotonated tridentate chelate H2L ligand (with NO2 donor sites) and one water molecule
and one nitrate ion. The geometry of the penta-coordinated metal center is measured by the
Addison parameter (τ), which is 0.006 for 1 in this case [τ = (α - β)/60, where α and β are the
two largest Ligand-Metal-Ligand angles of the coordination sphere], suggesting a nearly
square pyramidal geometry of around the Al3+ center (τ = 0 for a perfect square pyramid and
τ = 1 for a perfect trigonal bi-pyramidal). The equatorial plane is occupied by one amide enol
and one phenoxido oxygen (O1 and O2) and one immine Nitrogen (N1) and one water
molecules (O1w), while the one axial site is occupied by an oxygen atom (O1n) of nitrate ion
in 1. In 1, all calculated Al-N distances occur in the range 1.956Å and Al-O distances are in
the range 1.908-1.929Å.
On the other hand for complex 2, the metal center Cr3+ is being hexa-coordinated with one
di-deprotonated tridentate chelate ligand (with NO2 donor sites) and three water molecules to
form a distorted octahedral geometry. The calculated Cr–N bond distance is 1.861Å and Cr–
O bond distance are fall in the range 1.790-1.818Å.The equatorial sites are occupied by one
amide enol and one phenoxido oxygen (O1 and O2) and one immine Nitrogen (N1) and
oxygen (O3w) of the water molecule, while the axial sites are occupied by two aqua oxygens
(O1w and O2w) in 2.
In case of complex 1 at ground state, the electron density at HOMO, HOMO–1 and
HOMO – 2 orbitals are mainly originating from ligand π and π* orbital contribution while the
HOMO–3, LUMO and LUMO+1 orbitals arises from metal d orbital contribution along with
ligand π orbital contribution. The energy difference between HOMO and LUMO is 0.878eV
of ligand (H2L). In case of complex 2, the electron density at HOMO, HOMO – 1, and
HOMO – 2 orbitals are mainly resided on the benzene moiety and from metal d orbital
contribution while a considerable contribution comes from p-nitro azo-benzene moiety along
with the combination of rhodamine moiety in LUMO and LUMO+1 orbitals. The energy
difference between HOMO and LUMO is 5.015eV of 2.
The complex 1 shows a sharp absorption band at 554 nm in EtOH solution at room
temperature. This experimental value was supported by the TDDFT calculation. This
absorption band is assigned to the S0 →S26 transition which is in good agreement with
21

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experimental results of 547 nm. The absorption energies along with their oscillator strengths,
the main configurations and their assignments calculated using TDDFT method using the
ground state geometry for complex 1 is discussed here and the related data are given in Table
3.
The complex 2 exhibits one absorption band at 557 nm in ethanol solution at room
temperature. The TDDFT calculated absorption band is located at 553 nm for 2, which is in
good agreement with the experimental results of 557 nm (Table 4). This absorption band is
assigned to the S0 → S24 transition (Table 4).
Fig. 11. DFT/B3LYP optimized geometry of complexes [Al(L)(H2O)(NO3)]+ (1) and as well as
[Cr(L)(H2O)3]2+(2).
Table 1: Selected optimized geometrical parameters for [Al(L)(H2O)(NO3)]+ (1) in the
ground state calculated at B3LYP Levels.
Bonds
Al1-N1
Al1-O1
Al1-O2
Al1-O1w
Al1-O1n
Values (Å)
1.956
Bond angles
N1-Al1-O1
Values (°)
79.52
1.918
O1-Al1-O2
147.50
92.93
1.929
N1-Al1-O2
1.909
N1-Al1-O1n
N1- Al1-O1w
O1- Al1-O1w
O1- Al1-O1n
106.24
147.12
86.24
1.908
105.47
22

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O2- Al1-O1n
O2- Al1-O1w
O1a- Al1-O1m
O1a- Al1-O1w
O1a- Al1-O1n
O1m- Al1-O1w
O1m- Al1-O1n
106.97
83.34
88.75
90.21
90.07
173.77
84.92
Table 2: Selective bond distance and bond angles of [Cr(L)(H2O)3]2+ (2).
Bonds
Cr1-N1
Values (Å)
1.861
Bond angles
N1-Cr1-O1
Values (°)
84.32
163.35
99.31
84.57
95.43
167.81
179.97
83.27
98.41
98.07
81.91
81.52
96.70
96.50
83.39
Cr1-O1
Cr1-O2
1.818
O1- Cr1-O2
1.806
N1- Cr1-O2
Cr1-O1w
Cr1-O2w
1.806
1.790
N1- Cr1-O1w
N1- Cr1-O2w
N1- Cr1-O3w
O1w- Cr1-O2w
O1w- Cr1-O3w
O1w- Cr1-O1
O2- Cr1-O1w
O2w- Cr1-O2
O2w- Cr1-O1
O2w- Cr1-O3w
O3w- Cr1-O1
O3w- Cr1-O2
Cr1-O3w
1.800
23

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Fig. 12. Frontier molecular orbitals of optimized complexes.
Table 3: Selected parameters for the vertical excitation (UV-Vis absorptions) of complex 1 in
terms of molecular orbital contribution of the transition; electronic excitation energies (eV)
and oscillator strengths (f) in ethanol.
Electronic Composition
transition
Excitation Oscillator CI
λexp (nm)
energy
strength
(f)
S0 → S26
HOMO →LUMO + 1
2.5414 eV 0.0472
(547nm)
0.66278 554
HOMO – 2→ LUMO
0.63547
0.28628
HOMO – 1→ LUMO +1
24

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Fig. 13. Frontier molecular orbitals involved in the UV-Vis absorption of complex 1.
Table 4: Main calculated optical transition for the complex 2 with the composition in terms
of molecular orbital contribution of the transition, vertical excitation energies, and oscillator
strength in ethanol.
Electronic Composition
transition
Excitation Oscillator CI
λexp (nm)
energy
strength
(f)
S0 → S24
HOMO – 1 →LUMO
2.3627 eV 0.0309
(553 nm)
0.58251 557
HOMO →LUMO
0.27015
0.45910
HOMO – 2 →LUMO +1
25

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Fig. 14. Frontier molecular orbitals involved in the UV-Vis absorption of complex 2.
3.10 Time-Correlated Single Photon Counting (TCSPC) Study
The enhancement of fluorescence of L upon binding with Al3+ and Cr3+has been
supported by the results obtained from fluorescence decay measurements, using the TCSPC
technique (Fig. 15). The decay behavior of the bare probe and its metal complex is best fitted
to bi-exponential functions. Bare L showed two components having lifetimes 0.13 and 2.34
ns respectively. The populations of the same were 0.94 and 0.06 % respectively. The average
lifetime of L was calculated to be 0.54 ns. Upon addition of Al3+, two components were
obtained having lifetimes 0.38 and 1.89 ns respectively, having populations of 0.78 and 0.22
% respectively (Table 5). The average lifetime was calculated to be 1.31 ns. A similar
experiment performed with comparable Cr3+ showed a bi-exponential decay trace with time
constants τ1 = 0.36 (0.77 %) and τ2 = 1.72 (0.23 %). The average lifetime was calculated to
be 1.19 ns. Upon binding of Al3+ and Cr3+ to the (L) formation of a tight binding complex
occurs along with the opening of the spirolactam ring to convert the free (hanging)
Rhodamine B part of the complex, thereby freezing of non-radiative pathways generated by
26

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rigid structure of the complex than the comparatively more flexible structure of the bare
ligand.[62]As a result the average lifetime increased.
Table 5: Fluorescence lifetimes of L, L-Al3+ and L-Cr3+ complexes in EtOH-H2O solvent
EtOH/H2O
τ1(ns)
0.13
0.38
0.36
τ2(ns)
2.34
1.89
1.72
α1
α2
χ2
τav
0.94
0.78
0.77
0.06 1.07318 0.54
0.22 0.99236 1.31
L
L-Al3+
L-Cr3+
0.23
0.995
1.19
Prompt
Decay L
Decay L-Al3+
Decay L-Cr3+
1000
100
10
30
40
50
Lifetime (ns)
Fig. 15. Life-time measurements of L , L–Al3+ and L–Cr3+(λex = 520 nm)
3.11 Cell imaging study
The overnight sub-confluent culture of human lymphocyte cells (HLC) was washed with
phosphate buffer saline (PBS; pH 7.4) and incubated with DMEM containing L with the final
concentration at 5 µM for 3h at 37 °C in CO2 incubator. After incubation, fluorescence
images of HLCs were captured under a fluorescence microscope (LEICA DFC295, Germany)
in 40X magnification. Similarly, a fluorescence image of HLCs (pre-incubated with 5 µML)
was taken after addition of Al3+ and Cr3+ salt solution at the concentration of 10 µM for 1h
separately with an excitation wavelength 582 and 581 nm [63].
3.11.1 In vitro cell cytotoxicity
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To reproduce the capability of the fluorescence-based probe for intracellular imaging of Al3+
and Cr3+, firstly, it was of prime importance to verify the cytotoxic effect of the ligand on
normal human cells. To establish the biocompatibility of the ligand in the cellular
environment, the performed cell viability study by standard MTT assay in human lymphocyte
cells (HLCs) revealed that 93.6 ± 0.54 % cell viability was observed at the concentration of 5
µM in the presence of L compared to HLC-control cells. The viability of the cells has been
observed in a dose-dependent manner up to the concentration of 100 µM. Ligand showed
45.3 ± 0.48 % cytotoxicity at 100 µM concentration after 24 h incubation, whereas only 6.2 +
0.51 % cytotoxicity at 5 µM concentration at the same incubation time. It has been assumed
that ligand did not influence the viability of HLCs at least 24h of its treatment at
concentration 5 µM (Fig. 16).
Fig. 16. % cells viability study of HLCs treated with different concentration (5-100 µM) of
Ligand for 24h by MTT assay. Values are expressed as the mean ± S.D. of three
independent experiments.
3.11. 2 Fluorescence cell imaging study
No intracellular fluorescence was observed in a fluorescence cell imaging study, after the
incubation with the ligand at a concentration of 5 µM at 37 °C for 1h. However, intensive
intracellular green switch-on fluorescence is observed after the incubation with exogenous
Al3+ ion (10 μM) and Cr3+ (10 μM) solution and the fluorescence intensity of L-Al3+ is quite
higher than the L-Cr3+ salt solution (Fig. 17). The fluorescence image of the L with Al3+ and
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Cr3+ salt offers the confirmatory evidence that the L easily infiltrates the cell membrane and
bind with intracellular Al3+ and Cr3+ forming the L-Al3+ and L-Cr3+complex respectively.
The present study proposes that L could be utilized as an ineffaceable signature of the
selective sensor for bio-imaging Al3+ ions at the specified doses and incubation time without
showing any cytotoxic effect.
Fig. 17. (a) Fluorescence image of HLCs incubated with Ligand (5µM), (b) Al3+ salt solution
(10µM), and (c) Cr3+ salt solution (10 µM).
4. Conclusion
In summary, we have demonstrated an inexpensive, solvatochromic–based highly
efficient, fast turn-on probe, L. The L in its stable metal-induced conjugated chelated form
expresses its strong off-on selectivity response towards Al3+, Cr3+, and Cu2+ detected by UV-
Visible absorption whereas Al3+ and Cr3+ detected by fluorimetric study. Larger association
constants 6.7435×103 M-1, 3.8×103 M-1, and 5.685×103 M-1 for Al3+, Cr3+, and Cu2+,
respectively and nanomolar limit of detection 1.07×10-6 M, 2.02×10-6 M, 1.6×10-6 M for Al3+,
Cr3+, and Cu2+, respectively along with insignificant interference from the common co-
existing metal ions makes L as a convenient tool for effective detection Al3+, Cr3+, and Cu2+.
Our work is the first to report on Cr3+ chemosensor based on rhodamine-azobenzene moiety.
We have also constructed INHIBIT molecular Logic gate using the sensing properties. High
sustainability in physiological pH and high-resolution cell imaging experiment in human
lymphocyte cell further confirmed its applicability without toxicity in living biological
system for monitoring the intercellular level of Al3+ and Cr3+. We hope our rhodamine-
azobenzene based chemosensor will open up new possibilities for synthesizing cost-effective
dual analyte recognizing sensor in recent future.
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