A cell-compatible red light-emitting multianalyte chemosensor via three birds, one stone strategy

Article abstract of DOI:10.1016/j.jphotochem.2020.112889

The design and synthesis of red light-emitting multianalyte chemosensors have always been a challenging task because of its specific requirement of coordination pocket and selective fluorescence mechanism. Herein, we develop a chemosensor via “three birds

Full text of DOI:10.1016/j.jphotochem.2020.112889

Journal Pre-proof
A cell-compatible red light-emitting multianalyte chemosensor via three
birds, one stone strategy
Subhabrata Mabhai, Malay Dolai, Surya Kanta Dey, Anamika
Dhara, Sujata Maiti Choudhury, Bhriguram Das, Satyajit Dey, Atanu
Jana, Deb Ranjan Banerjee
PII:
S1010-6030(20)30686-9
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112889
JPC 112889
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
14 June 2020
28 August 2020
2 September 2020
Please cite this article as: Mabhai S, Dolai M, Dey SK, Dhara A, Choudhury SM, Das B, Dey
S, Jana A, Banerjee DR, A cell-compatible red light-emitting multianalyte chemosensor via
three birds, one stone strategy, Journal of Photochemistry and amp; Photobiology, A:
Chemistry (2020), doi: https://doi.org/10.1016/j.jphotochem.2020.112889
This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.
© 2020 Published by Elsevier.

1
A cell-compatible red light-emitting multianalyte chemosensor via three birds, one stone
strategy
Subhabrata Mabhai,a,f,h Malay Dolai,b Surya Kanta Dey,c Anamika Dhara,d Sujata Maiti
Choudhury, c Bhriguram Das,e,f Satyajit Dey,*f Atanu Jana,*g Deb Ranjan Banerjee*h
aDepartment of Chemistry, Mahishadal Raj College, East Midnapore, Mahishadal, West Ben-
gal, Pin No. 721628, India.
bDepartment of Chemistry, Prabhat Kumar College, Contai, Purba Medinipur 721401, India.
cDepartment of Human Physiology with Community Health; Vidyasagar University, Rang-
amati, Medinipur, West Bengal, Pin No. 721102, India.
dDepartment of Chemistry, Jadavpur University, Raja S. C. Mallick Road, Kolkata 700032,
India.
eDepartment of Chemistry and Chemical Technology, Vidyasagar University, Rangamati, Me-
dinipur, West Bengal, Pin No. 721102, India.
fDepartment of Chemistry, Tamralipta Mahavidyalaya, East Midnapore, West Bengal, Pin No.
721636, India.
gDivision of Physics and Semiconductor Science, Dongguk University, Seoul, 04620, South
Korea.
hDepartment of Chemistry, National Institute of Technology, Durgapur, W.B. PIN- 713209
Corresponding author.
Email addresses:
satyajitdeyoc@gmail.com (S.D.),
atanujanaic@gmail.com or atanujana@dongguk.edu (A.J.),
debranjan.banerjee@ch.nitdgp.ac.in (D.R.B.)
1

2
HIGHLIGHTS


A new PET – CHEF controlled rhodamine-azobenzene based chemosensor was reported.
Chemoselective detection of Cu2+, Al3+, and Cr3+ by UV-Visible absorption study and Al3+ and
Cr3+ by fluorimetric study

The detection limit is as low as 1.1 × 10−6 M, 1.3 × 10−6 M, and 1.5 × 10−6 M for Cu2+,
Al3+, and Cr3+ respectively


The 1:1 stoichiometry of the complex was confirmed by Job’s plot.
The sensor is capable to mimic an AND LOGIC GATE and can be used as onsight solid probe
(paper strip).

The sensor L is cell-permeable and is utilized to map the intracellular concentration of Al3+ and
Cr3+ in HLCs.
Abstract
The design and synthesis of red light-emitting multianalyte chemosensors have always
been a challenging task because of its specific requirement of coordination pocket and selective
fluorescence mechanism. Herein, we develop a chemosensor via “three birds, one stone” strat-
egy in which we can detect three metal ions with one ligand. A highly sensitive new azo func-
tionalized rhodamine based luminescent sensor is synthesized for selective fluorogenic recog-
nition of Al3+, Cr3+, and chromogenic recognition of Cu2+ in ethanol : H2O medium in the red
light-emitting zone. Among the guest metals, Cu2+ efficiently quenches the emission whereas
Al3+ and Cr3+ induce increased luminescent 4.76 fold for Al3+ and 2.47 fold for Cr3+ through
chelation-enhanced fluorescence (CHEF) and photo-induced electron transfer (PET) regulated
mechanism with the formation of 1:1 complex. The restricted imine isomerization through
complex formation inhibits ongoing PET process with the instantaneous onset of CHEF. The
13
mechanism is in good consonance with NMR (1H & C), FT-IR, elemental analysis, DFT,
TCSPC, and pH-dependent studies. Micromolar range detection of 1.1 µM, 1.3 µM, and 1.5
µM for Cu2+, Al3+, and Cr3+ respectively, easy penetration into HLCs cells and higher imaging
resolution increase its potentiality to assess Al3+ and Cr3+ in vitro. Moreover, paper strip appli-
cation increases its viability as an onsite naked-eye portable solid probe.
Keywords: Rhodamine, Azobenzene, Chemosensor, PET – CHEF, Paramagnetic quenching
2

3
1. Introduction
Besides natural sources, various industrial activities and anthropogenic activity enrich
the amount of Cu2+, Al3+, and Cr3+ in the environment. A living body requires a very low con-
centration of Cu2+. Its toxicity through excessive accumulation brings the loss of activity in
various Cu-dependent enzymes viz. tyrosinase co-oxidase, cytochrome, and superoxide dis-
mutase which is responsible for the generation of in vivo reactive oxygen regulation by iron
and causes Menkes disease by disturbing the development of the brain [1]. Wilson’s disease, a
Cu2+ related disease, is grown up by the excessive deposition of Cu2+ in the liver and brain
through irregular transport [2]. Among the different oxidation state trace element chromium is
less harmful in its trivalent (Cr3+) state [3]. Balance concentration of chromium regulates the
metabolism of fat lipids and carbohydrates by activating certain enzymes whereas insufficient
dietary intake increases blood sugar by deactivating insulin, increases the risk of cardiovascular
disease, and stops the formation of haemoglobin in the red blood cell [4]. Again diversified
application of alumina includes not only industries like packaging, pharmaceuticals, food, and
medicine but also in our daily life through cooking utensils, electronic gadgets, and perfumes.
Consumption of Al3+ over 3 – 10 mg per day has been linked to serious health disorders [5,6].
Over-exposure of Al3+ through acid rain and industrial practices increases the risk of competi-
tive inhibition of metal ions (Mg2+, Ca2+ and Fe2+ ) in the human body which is similar in size
,
and charge as that of Al3+ [7]. Aluminium related bone diseases like osteomalacia some neu-
rotoxic disease the Alzheimer’s and Parkinson’s disease suspected to be caused by overexpo-
sure of Al3+ [8].
Modern life cannot be circumvented without these essential metal ions even there re-
mains the risk of serious environmental and biological malfunctioning. Therefore there is a
constant demand for making ‘rapid’ and “simple-to-use” artificial optical receptors for easy
“Turn-on” detection of these trace elements in the nanomolar level [9,10]. Among all other
sensory systems swift sensitivity, selectivity, easy fabricability, and reusability continuously
increase the importance of cation sensing “Turn-on” fluorescent sensors over other probes in
various fields [11]. Our group encounters all the difficulties by developing a highly advanta-
geous single probe for detecting multiple analytes over one-to-one normal sensors in ethanol :
H2O medium [12–14]. Rhodamine framework, an automatic choice for an ideal “Turn-on” flu-
orescent platform for the “chemosensing ensemble” of cations not only because of its excellent
photostability and higher extinction coefficients (>75000 cm−1 M−1) but also it provides an
easily understandable model by toggling between spirocyclic nonfluorescent form and the
3

4
shiny fluorescent metal bounded ring-opening zwitterionic form [15]. Rhodamine groups of het-
erocyclic azo dyes are the perfect library of host for building up Off/On chemosensor by accepting a
large number of metal ions through the formation of stable chelate with the appreciable number of
metals through appropriate coordinating sites [16]. Azo dyes with higher molar extinction coefficient
have their significant contribution in the field of biological-medical studies as well as textiles, food,
cosmetics, and dyes - pigments industries [17,18].
Herein, we develop an azo dye-based Schiff base chemosensor L via “three birds, one
stone” strategy. L was synthesized by the condensation between Rhodamine B hydrazide and
(E)-2-hydroxy-5-(naphthalene-1-yldiazenyl) benzaldehyde. The L exhibits excellent photo-
chromatic behavior in ethanol: H2O system with the typical “Off–ON” method. Multiple sen-
sor–analyte recognition at physiological conditions, biocompatibility and mimicking ability of
advanced arithmetic logic operation within a combinatorial logic circuit make this an effective
sensor for recognizing Al3+ along with to date very limited reported paramagnetic Cu2+ (d9)
and Cr3+ (d3) through CHEF process [19]. Chemosensor for the detection of paramagnetic Cr3+
ion is rarely available. The L exhibits red fluorescence upon binding with the desired metal
ions. All the optical properties have been supported by DFT calculations. The potential appli-
cation of L in in vitro fluorescence imaging was effectively demonstrated using Al3+ and Cr3+
ions.
2. Experimental
2.1. General information for Chemicals and instruments
Unless specified in other way all analytical grade reagent salicylaldehyde (98 %), 1–
Naphthyl amine (96 %), Rhodamine B (98.0 %) from Merck - India, high-purity HEPES (98.0
%), Na2EDTA (97.0 %), and dry solvents were purchased commercially and were used without
further purification. Distilled water was used to prepare the stock solution of chlorides and
nitrates salts of the competitive cations (Na+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Cr3+, Fe3+,
Mn2+, Mg2+, Ni2+, Pd2+, Pb2+, Zn2+, Hg2+) purchased from SRL (India) Ltd. Thermo Evolution
(Model-201) and JASCO-8500 spectrophotometer were used to monitor UV – Vis and fluores-
cence spectra, respectively. Both spectroscopic analysis was performed in the quartz cuvette
(path length = 1 cm) with HPLC grade solvent at room temperature. Perkin–Elmer 2400 CHN
elemental analyzer analyzes the C, H, N, and IR spectra (KBr pellet, 400–4000 cm−1) was
recorded using Perkin-Elmer infrared spectrophotometer (Model: 883). Qtof Micro YA263
mass spectrometer measured the Electrospray ionization MS (ESI-MS). The Time-Correlated
4

5
Single Photon Counting (TCSPC) data runs on a HORIBA Jobin Yvon Fluorocube-01-NL flu-
orescence lifetime spectrometer. Photoluminescence quantum yield (FL QY) was determined
with the help of optically matching standard Fluorescein (Φr = 0.79). DFT calculation data was
collected by taking the help of Gaussian software. Brucker 400 MHz instrument was used to
record 1H and 100 MHz for 13C in DMSO-d6 at room temperature. Relative to TMS chemical
shifts were expressed in ppm and 1H-1H coupling constants in Hz.
2.2. Computational details
The calculation of ligand electronic structures in ground-state, complexes 1 and 2 in the
gas phase have been finalized by density functional theory (DFT) [20] method accompanying
the conductor-like polarizable continuum model (CPCM) [21–23]. The study was supported
by Becke’s hybrid function [24] with the Lee-Yang-Parr (LYP) correlation function [25]. The
spectral absorbance properties in the ethanolic medium for L, [Al(L)(H2O)]2+(1) and
[Cr(L)(H2O)]2+(2), were calculated using time-dependent density functional theory (TDDFT)
[26–28] associated with the CPCM model and we computed the lowest 40 singlet – singlet
transition. We are setting a 6-31(g) basis set for H atoms, and 6-31+g as the basis set for C, N,
O, Al and Cr atoms. Gauss View 5.1 software was used to make the calculated electron-density
plots for frontier molecular orbitals. All the computations have been carried on with the Gauss-
ian 09W programming software package [29]. Contributions of molecular orbital from groups
or atoms have been computed by using Gauss Sum 2.1 program [30].
2.3. Measurements of fluorescence lifetime
HORIBA Jobin Yvon Fluorocube-01-NL fluorescence lifetime spectrometer was used
to measure fluorescence lifetimes of all the samples. A laser diode of 450 nm excited the liquid
samples and the signaling responses were obtained at a magic angle of 54.7° to remove any
major inputs from anisotropy decay of fluorescence [31]. The standard time resolution for our
experimental setup is ~ 100 ps. The following equation is used to calculate the mean (average)
fluorescence lifetimes [32,33].
  2
 


i
i
av 
i
i
in which αi is the pre-exponential factor corresponding to the ith decay time constant, τi.
2.4. Procedure for Isolation of Human lymphocyte cell (HLCs)
5

6
From healthy young volunteers, blood samples (5 ml) were collected in 5 ml heparin-
coated Vacutainers Hudson and Hay [34] and an exact amount of blood (4 ml) were mixed
with the same volume of Histopaque 1077 (Sigma-Aldrich Co. LLC, US) followed by
centrifugation at 2000 rpm for 30 min to make an upper layer of blood at room temperature.
The upper lymphocytes monolayer was passed on to a sterile centrifuge tube followed by three
times washing by phosphate buffer solution at pH 7.4. Re-suspension of HLCs was performed
in RPMI media containing 10 % FBS and then incubated in a CO2 incubator for 24 h at 37 ºC.
2.5. Cytotoxicity study
Cytotoxicity study of L in HLCs cells was performed by MTT assay [34]. In brief, in 96-
well plates in 100 μL of RPMI medium, HLCs were seeded at a density of 1 × 106 cells per
well and treated with a ligand at a series of concentrations (5 µM, 10 µM, 20 µLM, 50 µM,
and 100 µM) for 24 h. The cell was washed thoroughly by PBS (pH 7.4) after removal. Each well
containing 10 µL of a stock MTT solution (1 mg mL-1 in PBS) was incubated for 3 h at 37 oC. ELISA
ANALYZER (Bio-Radd Model 680) was used to measure the absorbance at 540 nm of inter-
cellular formazan crystal after adding 0.01 % DMSO at each well. We calculate Cell
cytotoxicity and cell viability. with the following equation.
% cell cytotoxicity =100 % - % cell viability
% cell viability = (OD of the sample)/(OD of control) × 100
2.6. Cell imaging study
Washed HLCs cells with PBS (pH 7.4) were incubated with RPMI culture medium containing
the Ligand maintaining the ultimate concentration at 5 µM for 3 h at 37 0C in a CO2 incubator.
Following the incubation, cells were washed with PBS (pH 7.4) in triplicate, and fluorescence
images of HLCs cells were examined through a fluorescence microscope (LEICA DFC2 95,
Germany). Likewise, after pre-incubation with 5 µM ligand, the fluorescence image of HLCs
cells was taken with the addition of 10 µM solution of Cr3+, and Al3+ at with an excitation
wavelength of 530 and 581 nm.
2.7. Synthesis of rhodamine B hydrazide (2-amino-3', 6'-bis(diethylamino)spiro[isoindo-
line-1,9'-xanthen]-3-one)
6

7
Rhodamine B hydrazide (2-amino-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one) was
obtained as a pink solid after removal of excess ethanol from the reaction of Rhodamine B and hydra-
zine hydrate in refluxing condition [35].
2.8. Synthesis and characterization of (E)-2-hydroxy-5-(naphthalen-1-yldiazenyl)benzal-
dehyde.
An acidic mixture containing 1-naphthylamine (5.86 gm, 0.04 mol) and HCl (30 %, 25
ml) was prepared by continuous stirring at 60 oC for half an hour. The resulting solution was
diazotized by the dropwise addition of NaNO2 (2.756 g, 0.04 mol) at 0 oC - 5 oC, and stirred
nearly half an hour. The diazotized pale pink color ice-cold solution was dropwise added to the
50 ml alkaline salicylaldehyde solution containing NaOH (1.6 g 0.04 mol) and Na2CO3 (3.18
g 0.03 mol) at the above cold temperature. After 12 hrs of stirring at room temperature, the
resulting dark brown product was collected utilizing Whatman-41 and reslurried by washing
with 10 % NaCl. The pH of the slurried solution was adjusted to (pH 4–5) by adding the re-
quired amount of conc. HCl. The purity of red-brown color azo-dye obtained after air-drying
was established by chromatography. The yield of the product was 90 %. M.P. 140 oC. 1HNMR:
(Fig. S1, ESI) [400 MHz, DMSO-d6, δ (ppm)] δ 10.40 (s, 1H, Ar-OH), 8.85 (s, 1H, Ar-CHO)
8.31 (s, 1H, Ar-H) 8.24 (d, 1H, J = 7.2 Hz, Ar-H), 8.11 (d, 1H, Ar-H, J = 6.4 Hz), 8.05 (d, 1H,
Ar-H, J = 6.8 Hz), 7.78 – 7.63 (5H, m, Naphthalene ring proton), 7.24 (d, 1H, J = 7.2 Hz,
13
Naphthalene ring proton). C NMR (Fig. S2, ESI) [100 MHz DMSO-d6, δ (ppm)]: 112.23,
119.01, 123.12, 123.24, 125.16, 126.31, 127.16, 127.66, 128.56, 130.12, 130.85, 131.69,
134.41, 163.95, and 191.06.
2.9. Synthesis and characterization of chemosensor L (3', 6'-bis(diethylamino)-2-(((E)-2-
hydroxy-5-(naphthalen-1-yldiazenyl)benzylidene)amino)spiro[isoindoline-1,9'-xanthen]-
3-one)
Chemosensor L was synthesized by the condensation of Rhodamine B hydrazide and (E)-2-
hydroxy-5-(naphthalen-1-yldiazenyl)benzaldehyde (Scheme 1). An ethanolic Rhodamine B
hydrazide (0.5 g, 1.81 mmol) solution was poured to the stirring solution of (E)-2-hydroxy-5-
(naphthalen-1-yldiazenyl)benzaldehyde (0.825 g, 1.81 mmol) dissolved in dry ethanol. After
refluxing for 6 hrs with subsequent concentration of filtrate (1/3 of its original) the reaction
mixture had thrown out a deep red colored solid upon cooling. Solid was filtered and repeatedly
washed with cold ethanol – ether (1:1). Yield ~85 %. M.P 202 oC. 1H NMR of probe L (Fig.
S3, ESI) [400 MHz, DMSO-d6, δ (ppm)] δ11.10 (1H, s, -OH), 9.36 (1H, s, –CH=N), 8.89 (1H,
d, J = 5.6 Hz), 8.11 (2H, d, J = 6.8 Hz), 8.06 (1H, d, J = 6.4 Hz), 7.97-7.94 (2H, m), 7.77-7.61
7

8
(7H, m), 7.15 (1H, d, J = 6 Hz ), 7.04(1H, d, J = 7.2 Hz), 6.45 (3H, d, J = 7.2 Hz), 6.35 (2H, d,
J = 7.2 Hz), 3.26 (8H, NCH2CH3), 1.02 (12H, t, J = 5.6 Hz, -NCH2CH3).
13C NMR (Fig. S4, ESI) [100 MHz DMSO-d6, δ (ppm)] :12.68, 44.11, 66.24, 92.89, 97.81,
100.48, 105.62., 106.91, 107.33, 108.58, 111.98, 123.45, 126.32, 127.17, 127.54, 128.17,
128.55, 129.44, 130.91, 131.49, 134.44, 146.23, 147.18, 147.50, 148.99, 151.23, 153.20,
153.39, 160.45, 163.86, 164.48. Elemental analysis (calcd. %) for C45H42N6O3: C, 75.61; H,
5.92; N, 11.76; O, 6.71; found (%): C, 75.58; H, 5.85; N, 11.15; O, 6.65 ESI-MS: (Fig.S5, ESI)
m/z calculated C43H43N7O6 [M+]:715.34, found 715.51.
Scheme 1. Synthetic scheme of L
2.10. Synthesis and characterization of L-Al3+ and L-Cr3+Complexes
Al(NO3)3.9H2O (0.200 g, 0.533 mmol) and CrCl3 (0.084 g, 0.533 mmol) were injected sepa-
rately in the stirring solution of L (0.381 g, 0.533 mmol) in acetonitrile. After overnight stirring
a deep maroon solid deposited in each case was filtered and washed with cold MeOH repeat-
edly. The complex thus prepared was dried.
1H NMR L-Al3+ (Fig. S6, ESI) [400 MHz, DMSO-d6, δ (ppm)] δ 9.47 (1H, s, –CH=N), 8.88
(1H, d, J = 6.4 Hz), 8.11(2H, d, J = 6.4 Hz), 8.06 (1H, d, J = 6.4 Hz), 7.95 (2H, t, J = 6 Hz),
7.79-7.60 (7H, m), 7.15 (1H, d, J = 6 Hz ), 7.03 (1H, d, J = 7.2 Hz), 6.45 (3H, d, J = 7.6 Hz),
6.36 (2H, d, J = 6.4 Hz), 3.26 (8H, -NCH2CH3), 1.01 (12H, t, J = 5.6 Hz, -NCH2CH3).
13C NMR L-Al3+ (Fig. S7, ESI) [100 MHz DMSO-d6, δ (ppm)] : 12.76, 44.13, 97.93, 108.68,
111.98, 115.64, 117.78, 120.90, 123.07, 123.43, 123.59, 124.45, 126.33, 126.95, 126.97,
127.18, 127.56, 128.18, 128.56, 129.41, 129.47, 130.90, 131.50, 134..43, 134.55, 146.23,
147.17, 151.20, 153.37, 160.43, 164.45. 13C NMR L-Cr3+ (Fig. S8, ESI) [100 MHz DMSO-
8

9
d6, δ (ppm)] :12.87, 44.17, 76.15, 84.08, 91.96, 97.82, 105.67, 108.49, 111.80, 111.98, 117.72,
123.64, 124.46, 127.26, 127.62, 129.45, 129.55, 129.60, 130.93, 131.56, 134.44, 146.17,
147.08, 148.99, 151.99, 152.99, 153.39, 160.52, 160.61 164.17. ESI-MS: (Fig.S9, S10, ESI)
m/z calculated for C45H42N6O3 [L+Al+H2O - H+]+: 759, found 759.06 and for C45H42N6O3
[L+Cr+ H2O] : 785.51 found 785.51.
2.11. Procedure for fluorescence quantum yield measurement
Considering Φ, A, F, η as quantum yield, absorbance, the integrated area under the
emission and refractive index of the solvents respectively. FL QY is calculated by integrating
the total area under the emission spectra with the optically matching standard fluorescein (Φr
= 0.79) at same excitation and maintaining the same wavelength in the absorbance spectra in
ethanol: H2O medium by the following equation [36-37].
2
A F s
r
s  r (
s )(
)
2
A F r
s
r
Subscript s and r represent substrate and standard fluorescein, respectively.
2.12. Calculation of binding ratio (Job's plot)
A series of 10 vials containing a total of 20 μM L–Mn+ solution in 2 ml ethanol : H2O
(9:1,v/v, 5 μM HEPES, pH 7.2) were prepared by taking receptor solution and metal solution
in regular reverse order at room temperature. Job’s continuous variation method by using the
information from UV–Vis titration is applied to determine the binding stoichiometry of L-Mn+
at a regular interval of 2 mins [37].
2.13. Sensing procedure for metal ions
To study the optical properties of the sensor, L a stock solution with 1× 10−3 M con-
centration was adjusted in ethanol-aqueous HEPES buffer (5 μM, pH 7.2, 9:1, v/v). Selectivity
investigation was performed with 100 μL of different metal ions (c = 1 × 10−2 M) solution
under investigation prepared from ethanol-deionized water in a quartz cuvette (path length, 1
cm) containing 2 ml ethanol and 40 μL stock solution. Both UV – Vis and fluorometric titration
were performed with the addition of increasing aliquots of the metal ions with a concentration
of 1 × 10−3 M prepared from the same ethanol-deionized water.
2.14. Association constant calculation
9

10
Association constant (Ka) of the L – Mn+ complex is calculated based on Benesi–Hil-
debrand (B-H) equations [38] by the linear fitting of absorbance and fluorescence titration data.
1
1
1


(A A ) [K(Amax  A )C] (Amax  A )
0
0
0
and
1
1
1


(I  I0 ) (I1  I0 ) (I1  I0 )k[M n ]m
Where in presence of excess metal ion during titration (48 µM Cu2+, 72 µM Al3+, and 80 µM
Cr3+) Amax represent maximum absorbance of L (20 μM) at λmax = 551, 554, and 558 nm for
Cu2+, Al3+, and Cr3+. A0 stands for the maximum absorbance of sensor L and A is the recorded
absorbance for different concentrations of the metal ion at that particular wavelength.
Similarly, at 540 nm for (20 μM) L I0, I, and I1 represents the maximum emission
intensities of L in the absence of, for the intermediate and infinite concentration of the metal
ion. [Mn+] represents the metal ion concentration in both equations. The association constant
(ka) 12.27 × 103 M-1, 13.6261 × 103 M-1, 33.56 × 103 M-1 for L-Cu2+, L-Al3+, and L-Cr3+ com-
plex respectively is determined from the ratio of slope ant intercept (Fig. S13, S14, S15, ESI).
The good linearity of the plot (A-A0)-1, as a function of the reciprocal molar concentration,
[Mn+]-1 along with 0.99 -1 correlation coefficient value (R2) for all the complex supports the
formation of 1:1 complex, An actual concentration of 20 × 10-6 mol L-1 ligand solution at pH
7.2 from ethanol : H2O medium and the metal ions with the concentration of 1 × 10-3 mol L-1
is prepared from aq. solution by maintaining the same pH for this experiment.
3. Results and discussion
3.1. Studies on selectivity
The optical discrimination of L over other biologically active metal ions through its
chelating ability is confirmed by visually as well as spectrometrically in 2 mL ethanol: H2O
(9:1, v/v, 5 μM HEPES pH 7.2) solvent containing 40 μL of L at room temperature. Almost
colorless free receptor shows an immediate response upon the immediate inclusion of Cu2+,
Al3+, and Cr3+, by changing its color from colorless to deep pink. The selectivity of the binding
pocket provided by the L is confirmed by the appearance of three new spectral band positions
at λmax = 551, 554, and 558 nm respectively for Cu2+, Al3+, and Cr3+ in the absorbance study
(Fig. 1). Although the L shows some color change with Mn2+ in ambient light along with Al3+,
10

11
Cr3+, and Cu2+ absorbance study shows complete sensitivity only with these three metals.
Moreover, under the exposer of 366 nm UV lamp, the L response rests only with Al3+, Cr3+
(Fig. 4a). In the emission profile, two new band position at λmax = 580 nm and 579 nm at
saturation point appears by obliterating the spectral line of the L under similar condition (Fig.
4b). Taking advantage of this selectivity our group exploits the L as a single probe for dual ion
responsive molecular sensor through emission study and analyze its potential as a bioimaging
sensor in the living cell. Sensor L with its hard nitrogen and oxygen-containing base centers
provides a suitable binding pocket for diamagnetic, oxophilic hard acid Al3+ and increases the
rigidity to induce emission by deactivating the non-radiative process in the excited state [39].
Although other metal ions of p block like Ca2+ and Ba2+ because of their hard acidity can also form
complex obeying SHAB (soft hard acid base) principle with hard donor centers of ligand but greater
stability of L-Al3+ as because of its high ionic potential with respect to rest metal ions refutes their
possibility of forming a strong complex with ligand in presence of Al3+ [40]. The hard chelating site of
probe also holds the Cr3+ effectively in presence of other metal ions for its hard-hard interaction with
N/O containing chelating site which is perfectly substantiated from its higher binding constant [4]. Par-
amagnetic d9 Cu2+ acts as a natural fluorescence quencher by generating a deactivating radia-
tive channel through the transfer of excitation energy from ligand to metal d orbital and also
promotes the intersystem absorption phenomenon by transferring the electron to the Rhoda-
mine – B fluorophore in the excited singlet state [41-43].
3.2 Photophysical study
3.2.1. Absorption studies
To appraise the selective detection of Cu2+, Al3+, and Cr3+ by the sensor L (20 µM) with
the intervention of various other metal cations (Na+, K+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+,
Cr3+, Fe3+, Mn2+, Mg2+, Ni2+, Pd2+, Pb2+, Zn2+, and Hg2+) quantitative titration in UV–Visible
absorption spectra was appropriated in the prefixed ethanol : H2O ligand solution (9:1, v/v) pH
= 7.2 using 5 μM HEPES buffer at 25 °C. To support the absorbance selectivity, which exposed
by Cu2+, Al3+, and Cr3+ with the alternation of ligand spectral line at λmax = 551, 554, and 558
nm (Fig. 2 (a-c)) reversible experiment between the chelating complex of L-Mn+ with
Na2EDTA was performed.
11

12
(a)
(a)
Lig
2.5
2.0
1.5
1.0
0.5
0.0
Al3+
Ba2+
Na+
K+
(b)
Cu2+
Ca2+
NI2+
Cd2+
Co3+
Cr3+
Fe3+
Hg2+
Mg2+
Pb2+
Zn2+
Mn2+
Cu2+
Al3+
Cr3+
450
500
550
600
650
Wavelength(nm)
Fig. 1. Absorption spectral responses of Probe L (20 µM) after addition of diverse metal ions
(Na+, k+ Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Cr3+, Fe3, Mn2+, Mg2+, Ni2+, Pd2+, Pb2+, Zn2+, Hg2+)
25 equiv. in ethanol : H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C. Inset: (a) Visual depiction
of L with metal ions under ambient light.
Addition of different concentration Cu2+ (0 – 48 μM), Al3+ (0 – 72 μM), and Cr3+ (0 –
92 μM) to sensor L (20 µM) generates three new bands at saturation point with the gradual
deepening of pink color. Although Rhodamine B is a potential fluorogenic agent shows no
band in the absence of appropriate visitor metal as a result of its stable closed spirolactam ring
[42]. The reversibility of complex was confirmed when the L-Mn+ complex loses its pink color
after the gradual addition of Na2EDTA (Fig. S11 (a-c) ESI). The UV–Visible titration data
were used for calculating the binding constant (Ka) and it was found to be 12.27 × 103 M-1,
13.62 × 103 M-1, 33.56 × 103 M-1 L-Cu2+, L-Al3+, and L-Cr3+ complex respectively (Fig. S13,
S14, S15, ESI). The calculated LOD value according to the IUPAC method was 1.1 × 10−6 M,
12

13
1.3 × 10−6 M, and 1.5 × 10−6 M for Cu2+, Al3+, and Cr3+ respectively which fits well the per-
missible value referred by WHO and also better than many recently reported Al3+, Cr3+ sensor
(Fig. S16, S17, S18, ESI). A comparison table of different sensors with our based on the de-
tection limit is given in (Table S2, ESI).
(c)
(b)0.6
(a)
2.5
2.0
1.5
1.0
0.5
0.0
1.2
3+
3+
72  Al
92 Cr
2+
48 Cu
1.0
0.8
0.6
0.4
0.2
0.0
0.4
0.2
3+
0 Cr
2+
3+
0 Cu
0 Al
450
500
550
600
450
500
550
600
450
600
Wav5e0l0ength(5n5m0 )
Wavelength( nm)
Wavelength(nm)
Fig. 2. Changes in the absorption profile induced by the titration of probe L (20 µM) with
(a) Al3+ (b) Cr3+(c) Cu2+ (c = 1.0×10-3 M) in ethanol: H2O [9:1, v/v, 5µM HEPES pH 7.2].
3.2.2. Fluorescence study
To understand the photophysical properties of sensor L, photoluminescence (FL) and the time-
resolved spectroscopic experiment was performed in an appropriate solvent. Different solvent
was investigated to get a better reaction media with maximum emissive intensities and in both
cases, ethanol was the fittest solvent (Fig. 3a). To avoid the unnatural environment and sup-
pression of sensor signal to analytes by H2O a high ratio of organic solvent was used and fi-
nally, it was confirmed that the highest fluorescent emission comes from organic co-solvent
ethanol: H2O in (9:1 v/v) (Fig 3 (b-c)) [44]. The luminescence trait of L above λex = 540 nm
was persued for chloride salts of different cations viz. Na+, k+ Al3+,Ba2+, Ca2+, Cd2+, Co2+, Cu2+,
Cr3+, Fe3, Mn2+, Mg2+, Ni2+, Pd2+, Pb2+, Zn2+, Hg2+ in above mentioned solvent system at pH =
7.2 using 5 μM HEPES buffer at 25 °C. The non-fluorescent sensor L changes its luminescent
property encouragingly with the gradual addition of Al3+ and Cr3+ only. The other co-existing
metal ions along with Cu2+ do not afford any emission signal upon the addition of 25 eq. each
metal ions separately to the sensor solution when the same experimental condition was pro-
vided (Fig. 4b).
So, we sorted our fluorescence titration only with Al3+ and Cr3+ to have a clear idea about the
reversibility and binding property of L. The non-emissive nature of L upon irradiation λex =
13

14
540 nm is mainly by maintaining a closed-shell spirolactam ring which becomes fluorescent
after the addition of Cr3+ and Al3+ due to the formation of opened spirolactam ring which onset
(b)
(c)
Fig. 3. (a) Bar plot of the emissive pattern of sensor L as a function of solvent change to-
wards Al3+and Cr3+, Inset: corresponding photographic illustration under 366 nm UV light;
top L-Al3+, bottom L-Cr3+. Image of different emission intensities (b) L-Al3+, and (c) L-
Cr3+ in various ethanol: H2O system. (Fl. Indicates fluorescence )
the chelation-enhanced fluorescence (CHEF) by arresting ongoing photoinduced electron
transfer process (PET) [45]. Because of the shifting of equilibrium towards L-Al3+ and L-Cr3+
complex, the emission peak at 580 nm for Al3+ and 579 nm for Cr3+ gradually grows up and
reaches the maximum upon addition of 56 μM Al3+ and 84 μM Cr3+ with a 5 fold and 2.42 fold
increment in quantum yield with respect to bare L, respectively [Fig. 5(a-b)] [46].
Disodium salt of ethylenediaminetetraacetate (Na2EDTA) was used as a recovery agent to test
the reversibility of L. By increasing the concentration of Na2EDTA a much stronger guest for
14

15
sensor L than Al3+ and Cr3+ in ethanol: H2O the intensity of the complex decreases to the almost
similar manner and at the end completely bleaches the color of the complex (Fig. S12 (a-b),
ESI). The stronger complexation for diamagnetic Al3+ by CHEF induced open spirolactam ring
is responsible for higher emission intensity and higher quantum yield for Al3+ than Cr3+.
(a)
(b)
2500
L
3+
Ca2+
Al
Cd2+
3+
Co2+
Cr
2000
Cu2+
Fe3+
Hg2+
Mg2+
1500
Na+
Pb2+
Zn2+
Al3+
1000
Cr3+
Ni2+
Mn2+
K+
500
Pd2+
0
560
580
600
620
640
Wavelength(nm)
Fig. 4. Changes in the fluorescence of Probe L (20 µM) induced by the addition of diverse
competitive metal ions (10 equiv.) in ethanol: H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C
(a) Photographs of fluorescence under 366 nm illumination. (b) Corresponding emission
spectra (λex = 540 nm).
15

16
(a)
2500
(b)
2000
1500
1000
500
0
3+
3+
3+
56 Al
88 Cr
2000
1500
1000
500
0
3+
0 Al
0 Cr
550
575
600
625
650
550
600
650
Wavelength(nm)
Wavelength(nm)
Fig. 5. Luminescent profile of the probe L (20 µM) during concomitant addition of (a) Al3+
and (b) Cr3+ in ethanol: H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C. Inset: Shifting of visual
color is observed with the addition of Al3+ and Cr3+ in the ethanol: H2O solution of probe L as
observed under UV light illumination (366 nm).
3.2.3. Interference test
Achieving the selectivity over a background of the co-existing metal ion is a challeng-
ing task for developing a good sensor. To investigate the practical applicability of sensor L as
a selective host interference studies were performed by adding other diverse metal ions in the
presence of L–Al3+ and L–Cr3+ in ethanol: H2O medium under the same experimental condi-
tion separately (Fig. 6). No obvious change is observed when 5 equiv. of competitive cations
were added as that of Al3+ and Cr3+ to the sensor L under identical condition but with the addi-
tion of 10 equiv. of Fe3+ and Cu2+, a remarkable quenching in emission intensity is noticed. The
above experimental fact is explained by considering the charge transfer effect which happens
when the metal ion Cu2+ (d9) encounters the sensor L in the excited state and decreases the
number of the emitted electron by acting as an electron-acceptor [46]. The dramatically fluo-
rescence quenching by the highly electrophilic (charge density of Fe3+ is 6.09) Fe3+ is due to
favorable photo-induced electron transfer in between larger π conjugated Rhodamine moiety
and paramagnetic Fe3+. Higher electron affinity of Fe3+ ensures the onset of the PET process
by transferring electron density from large conjugated organic fluorophore [47,48]. The out-
comes achieved indicates that synthesized sensor safely be used with high precision through
turn-on fluorescence for labelling Al3+ and Cr3+.
16

17
M = Al3+ / Cr3+
Fig. 6. Proportionate bar plot of emission intensity of L in the fluorescence study with 5
equivalents of competing metal ions as that of Al3+/Cr3+ under the matching condition in
ethanol: H2O [9:1, v/v, 5 µM HEPES pH 7.2] at 25 °C.
3.2.4. Reversibility test
Stability, reversibility, and reproducibility are three important features that increase the
practical usability of the sensor and at the same time decrease the analysis cost. The reversible
nature, as well as the fatigue resistance of the complex in the presence of a strong guest than
L–Mn+ under identical condition, is well understood by periodical modulation of the metal -
Na2EDTA concentration. Reversibility experiment exposed that without any apparent degra-
dation of emission at 540 nm photophysical property of sensor L toggle between two states:
the CHEF induced a highly fluorescent state of L-Mn+ complex and metal-free closed-spiro-
lactam shell system where the pink color is almost completely bleached. The reproducibility of
this experiment confirms the reversibility of L by altering the concentration of both metal and
EDTA stimuli (Fig. S19, ESI).
3.2.5. pH study
17

18
The stability of the probe in physiological pH is very important for biological applica-
tions. To avoid the demetallation from sensor L through hydrolysis we have used appropriate
HEPES buffer solution in ethanol: H2O throughout the sensing studies [4]. To verify the pH
stability of the sensor L equimolar mixture of different sets of L–Al3+ and L–Cr3+ ensemble
was verified over a broad range of pH (2-12). Dominating spirolactam ring in the metal-free
condition is the root cause of lacking any type of fluorescence emission within the 4-11 pH
range. At lower pH in high acidic condition, enhancement of fluorescence of sensor L is due
to proton-induced opened spirolactam ring [49]. Emission intensities of the complex peaked
up its maximum level at pH – 5 and maintain the value up to pH – 9 after that the emission
intensity drops sharply till it reaches a constant value over pH ≥ 11 due to rapid demetallation
and re-establishing the closed spirolactam shell in the alkaline condition. Considering the hy-
drolysis at higher pH range and proton-induced emission at lower pH and fair stability at phys-
iological pH the L–Mn+ ensemble is an obvious choice for labelling Al3+ and Cr3+ in the bio-
logical system at physiological pH (Fig. 7)
200
In absenceof analytes
3+
In presence of Al
3+
In presence of Cr
150
100
50
0
0
2
4
6
8
10
12
pH
Fig. 7. Influence of pH on fluorescence response of L-Al3+ (brown dashed line) at λmax = 580
nm, L-Cr3+ (red solid line) at λmax = 579 nm, and L (black dotted line) in ethanol: H2O [9:1,
v/v at pH = 7.2 HEPES buffer] at 25 °C at the excitation wavelength of 540 nm.
3.2.6. Supportive pieces of evidence for the complex forming mechanism
18

19
As expected “Turn-Off” property of sensor L with its lower quantum yield under the
excitation at 540 nm during fluorescence study is due to the cumulative effect of PET and non-
radiative quenching by C = N isomerization [50]. The photoinduced electron transfer in the
dominant spirolactam tautomer is mainly due to the displacement of HOMO electron from the
N center of spirolactam ring and Schiff base nitrogen and - OH group to the fluorophore LUMO
to arrest the emission [51,52]. However, appropriate co-ordination by specific metal ion with
the strong donor site of the binding pocket offered by the imine – N, hydroxyl - O, and the
oxygen emerges from opening closed Rhodamine core initiates the CHEF process seizing on-
going PET by engaging the non-bonded electron. Therefore, we rightly affirm that the complete
makeover of the non-fluorescent colorless sensor to a pink fluorescent sensor as a result of
induced rigidity created by efficient chelation which is accompanied by –C = N isomerization.
The strong response of L as a result of strong chelation with Al3+ and Cr3+ in the suitable bind-
1
13
ing pocket of the host is well documented through Job’s plot experiment, H - NMR, C -
NMR, Mass, and IR spectrometric study. The appearance of the highest point for the mole
fraction of 0.5 for both the complex through a carefully conducted Job’s plot experiment in
ethanol: H2O solution (9:1 v/v) proves the 1:1 binding (Fig. S20, S21, S22, ESI) stoichiometry
between L and Al3+ and Cr3+ which was well supplemented by the formation of two peaks in
HRMS-ESI spectra at 759 for [L + Al3+ + H2O - H+], and at 785.51 for [L + Cr3+ + H2O] (Fig.
S9, S10, ESI). Shifting of C = O frequency of Rhodamine molecule at 1709 cm-1 to lower
frequency 1631 cm-1 for L-Al3+ and at 1615 cm-1 for L-Cr3+ along with broadening of – OH
peak indicates the high polarisation of these bond as a result of good induction with guest metal
(Fig. S23, S24, S25, ESI) [53,54]. The ring-opening phenomena through chelation also get
support from 1H and 13C – NMR spectral study of L in the absence and presence of Al3+ and
Cr3+ in DMSO-d6 solution. As revealed in the recorded spectra that complexation with Al3+ and
Cr3+ to the host completely iron out the signal for – OH proton at (δ) 11.092 ppm and for tertiary
sp3 carbon at (δ) 66.24 in 1H and 13C – NMR spectra respectively with the downfield shift of
imine proton (∆δi = 0.11 ppm) in 1H NMR conspicuously supports the complexation process
through imine-N, hydroxyl-O and oxygen of –CONH from Rhodamine moiety (Fig. S3, S4,
S6, S7, S8, ESI). Considering the above supportive evidence, a probable 1:1 complexation of
sensor L with Al3+ and Cr3+ is proposed in scheme 2. From the theoretical part, it is confirmed
that energy gap between highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) in the metal-induced complex is smaller than the probe L which is
responsible for producing a bathochromic shift in the absorption profile and is supportive for
metal complexation [55]. Information about the average lifetime of both complexes from the
19

20
TCSPC experiment also supports the mechanism for turn-on sensing by 1:1 complexation.
From Table – 2 of the TCSPC experiment it is noticed that τav for both L-Al3+ (1.65 ns) and L–
Cr3+ (1.40 ns) is much higher than the free ligand and the information is very much conclusive
for the formation of better complexation. The Quantum yield of the ligand (Φ = 4.6 %) taken
in the absence, and presence of metal ion increases to 4.76 fold (Φ = 21.94 %) for Al3+ and
2.47 fold (Φ = 11.34 %) for Cr3+.
Scheme 2. (a) Schematic presentation of (i) PET (ii) CHEF mechanism involved in sensing
for Al3+ and Cr3+ by sensor L. (b) Graphical representation of (i) non–emissive and (ii) Emis-
sive behavior of sensor in presence of metal through CHEF and in absence of metal through
C = N isomerization. (c) Exposes the probable binding mode between sensor L and analytes.
20

21
3.2.7. Electronic structures by geometry optimization
The geometries of L and its Al3+ and Cr3+ complexes are optimized and shown in Fig.
8a. The confirmation of the complexes composition is confirmed from HRMS data and then
performed the theoretical geometry optimization on it. The valuable optimized geometrical
parameters of the complexes are recorded in Table 1.
Table 1: Important bond lengths and bond angles of 1 and 2
Bond distance (Å)
Bond angles (°)
Complex-1
Complex-1
Al1-O1
1.790 O1- Al1-N1
1.786 N1-Al1-O2
1.791 O1-Al1-O2
93.18
Al1-O2
109.34
124.73
Al1-O1w
Al1-N1
1.769 N1-Al1-O1w 119.30
Complex-2
O1-
Al1- 111.15
O1W
Cr1-O1
1.947 O2-
O1W
Al1- 100.61
Cr1-O2
Cr1-O1w
Cr1-N1
1.777 Complex-2
2.058 O1- Cr1-N1
1.817 N1-Cr1-O2
O1-Cr1-O2
85.20
98.92
137.37
N1-Cr1-O1w 124.86
O1-
Cr1- 108.34
O1W
O2-
Cr1- 103.72
O1W
For both complexes 1 and 2, the metal atoms Al3+ and Cr3+ are actually tetra-coordinated with
the tri-dentate ligand L having mono-deprotonated NO2 donors and only aqua molecule to at-
tain the arrangement of a distorted tetrahedral geometry to form mono-cationic complex
[Al(L)(H2O)]2+(1) and [Cr(L)(H2O)]2+(2) respectively. The calculated M(III)–N bond dis-
tances are span in the range 1.769-1.817 Å and M(III)–O bond distances are span in the range
1.777-2.058 Å.
21

22
Fig. 8. (a) Geometry optimized molecular structures of L, [Al(L)(H2O)]2+(1) and
[Cr(L)(H2O)]2+(2). (b) Frontier molecular orbital of L, [Al(L)(H2O)]2+(1) and
[Cr(L)(H2O)]2+(2).
In case of L, the electron density resides mainly on HOMO – 1, LUMO and LUMO +
2 orbital occur at the naphthyl ring and phenoxo oxygen, whereas the same for HOMO,
HOMO–2 and LUMO + 1 orbitals remain at the Rhodamine moiety and phenoxido oxygen
with an energy gap between HOMO and LUMO of 2.64 eV (Fig. 8b). In case of complex 1,
HOMO, HOMO – 1 and LUMO + 1 orbitals mainly originate from the contribution of azine
nitrogens, Rhodamine moiety, and phenoxido oxygen and also LUMO, LUMO + 3 and HOMO
– 2 orbitals originate from azine nitrogens, phenoxido oxygen and considerable contribution
from metal d orbital with an energy gap between HOMO and LUMO of 0.74 eV (Fig. 8b). In
case of complex 2, HOMO, HOMO – 1 and LUMO + 3 orbitals generally exist on the phenox-
ido oxygen and a significant part comes from Rhodamine moiety along with the mixture of
metal d orbital in HOMO-2, LUMO and LUMO+1 orbitals with an energy gap between HOMO
and LUMO of 0.67 eV (Fig. 8b) in the ground state. The absorbance spectra of the complexes
1 and 2 used in the present work were studied at room temperature in MeOH. Complex 1 shows
only one peak at 554 nm (Fig. S26. ESI) which is in good agreement with theoretical value at
551 nm. This band is assigned to S0S4 electronic transitions. On the other hand, the complex
2, shows one well-consigned absorbance band at 558 nm (Fig. S26. ESI) which corresponds
22

23
to the theoretical absorption band that is matched at 553 nm which is in well fit with experi-
mental results (Table 1 ESI). These spectra bands can be given to the S0S3 transition.
3.2.8. TCSPC Study
Impact of the complexion of Al3+ or Cr3+ to the receptor L on the excited state lifetime
was further scrutinized from the outcomes of the Time-Correlated Single Photon Counting
(TCSPC) studies using a 450 nm nano-LED as the excitation source (Fig. 9). The decay profile
of L and its complex counterpart exhibit triexponential decays to obtain reasonable χ2 values
and the results are summarized in Table 2. Receptor L decayed through three channels having
lifetimes 1.03, 4.10, and 0.07 ns and relative populations of 9 %, 2 %, and 89 %, respectively.
The mean lifetime of the receptor L was calculated as 0.68 ns.
Fig. 9. Time-resolved fluorescence decay profile of probe L in HEPES buffer only and with
added Al3+ and Cr3+ ions (λex = 450 nm) 25 °C using a nano LED of 450 nm as the light source.
Upon binding with Al3+, lifetimes of three components changed to 0.58, 1.34, and 0.29
ns with relative populations of 35 %, 44 %, and 21 %, respectively and the mean lifetime was
increased to 1.65 ns. An analogous experiment done with comparable Cr3+ revealed a triexpo-
nential decay with the time τ1 = 1.14 (51 %), τ2 = 3.36 (3 %) and τ3 = 0.34 (46 %). The decay
time related to the closed-shell spirolactam moiety for the excited state was assigned to the
minor and short-lived component while the xanthene form of the receptor L was recognized
23

24
for the long-lived major component. The comparative large population of the open-ring xan-
thene as a result of the addition of Al3+ or Cr3+ evidenced the strong interaction of Al3+ or Cr3+
with the receptor. As a consequence, the mean lifetime has been enhanced. The restriction
imposed by the complexation in the free motion of flexible bonds as a result of rigid structural
integration intensifies the fluorescence intensity and the lifetime of corresponding excited-state
minimizing the non-radiative transitions [56].
Table 2. Fluorescence lifetimes of L, L-Cr3+ and L-Al3+ complexes in EtOH solvent
Metha- τ1(ns)
τ2(ns)
τ3(ns)
α1
α2
α3
χ2
τav
nol
L
1.03
4.10
1.34
3.36
0.07
0.29
0.34
0.09
0.35
0.51
0.02
0.44
0.03
0.89
0.21
0.46
1.03
1.07
0.99
0.68
1.65
1.40
L-Al3+ 0.58
L-Cr3+ 1.14
3.2.9. Boolean Logic Operations
The interesting chromo fluorogenic character exhibits by the sensor L through inde-
pendent modulation of Al3+, Cr3+, and EDTA inspired us to construct a combinational molec-
ular logic gate circuit by molecular interplay. This will give a clear idea about their photophys-
ical properties when they co-exist. The noncumulative type logic gate was framed using three
inputs: Al3+ (Input 1) Cr3+ (Input 2) and EDTA as input 3 and output as maximum fluorescence
emission for Al3+and Cr3+ at λem = 580 nm and 579 nm. In order, to mimic the advanced logic
operation binary digits (1 and 0) are used. Where 1 represents the signal on i.e. ‘YES’ state and
0 represents signal Off i.e. ‘NO’ state. When input 1 and input 2 are dropped, the output results
in Zero which represents the ‘OFF’ state because of the presence of non-fluorescent closed
spirolactam core. In presence input 1 and input 2 the output is maximum representing the 1 or
‘ON’ state, while in the presence of input 3 i.e. EDTA the output signal is always 0 indicating
‘OFF’ state. Therefore by judging all the strings of 16 truth tables, our combinational logic gate
supports the AND gate integrating OR and NOT gate which is portrayed in the [Fig. 10 (a-b)]
[57].
3.3.0. Development of L as a solid probe for Al3+ and Cr3+
To validate the practical relevancy of L for convenient onsite analysis as a solid probe
by exploiting its conspicuous color changing ability in the companionship of Al3+ and Cr3+ we
24

25
performed a simple paper strip examination. For this purpose, a TLC (thin layer chromatog-
raphy) plate was prepared by spotting an alternating solution of 16 metal ions on the ligand
spot. The experiment shows the obvious color change for Mn2+, Cu2+, Cr3+, and Al3+ under
ambient light and only Cr3+, and Al3+ under 366 nm UV light (Fig. 10 (c-d)) confirming the
formation of stable L–Mn+ complexation for Al3+ and Cr3+. Therefore, the probe can be used
as a valuable practical sensor to detect Cr3+, and Al3+ in the naked eye in solid-state.
(a)
(b)
INPUTS
In 2
OUTPUTS
Fl. Inten-
sity (a.u.)
In 1
In 3
EDTA
Al3+
Cr3+
580/579
nm
0
1
1
1
0
0
0
0
0
0
1
1
0
0
1
1
0
1
0
1
0
1
0
1
0
0
0
0
1
1
1
1
(c)
(d)
Fig. 10. (a) A Truth table for AND Logic gate. (b) Diagram for AND Logic Gate using tradi-
tional gate characters. (c) TLC paper strip in ambient light. (d) The TLC paper strip under 366
nm UV light.
3.3.1. Cell studies
In a comprehensive Cell viability experiment in HLCs cells, the cell viability of 92.02
± 0.37 % at 1 µM concentration is observed by MTT assay in the presence of ligand in contrast
to HLCs control cells. Cell viability has been examined in a sequential additive manner up to
100 µM. At the concentration of 100 µM L maximally showed 55.22 ± 0.39 % cytotoxicity
25

26
whereas only 3.07 + 0.48 % and 7.98 + 0.43 % cytotoxicity at 0.5 µM and 1 µM concentration
respectively. Therefore, from the above viability experiment unequivocally it can be inferred
that at the 0.5 µM and 1 µM concentration level at least for 24 hrs the viability of HLCs cell is
not influenced by the ligand (Fig. 11(a-b)).
In fluorescence cells imaging studies, no intracellular fluorescence was observed
following the nesting of Ligand L at 1 µM concentration of at 37 0C at least for 3 h. However,
cells display typical red fluorescence when Al3+ and Cr3+ solutions were injected to it and
obvious higher intensity of Al3+ in comparison to Cr3+ supports the better complexation of the
ligand with Al3+ in HLCs cell (Fig. 11c). This result indicates that during the incubation period
ligand penetrates the cell membrane and forms a strong complex with intracellular Al3+ salt.
The present study highlighted the non-toxic nature of the ligand which might be assigned as an
ineffaceable signature for specific in-vivo bio-imaging of Al3+ ions .
Fig. 11. (a - b) % cell viability study of HLCs cells conducted with various concentrations
(0.5-100 µM) of L for 24 h through MTT assay. Values are indicated as the mean ± S.D. of
three separate independent experiments. (c) luminescence microscopy image of HLCs cells
26

27
nurtured with [(A) L (1 µM) only (B) Cr3+ salt solution (10 µM) with L, and (C) Al3+ salt so-
lution (10 µM) with L.
4. Conclusion
In summary, we have described a red light-emitting multianalyte chemosensor follow-
ing “three birds, one stone strategy” for the detection of Cu2+, Al3+, and Cr3+ through UV–
Visible spectral study and Al3+, and Cr3+ by the fluorometric study in ethanol: H2O medium.
The sensor L through ring-opening approach forms a very stable metal-induced chelate com-
plex for Cu2+, Al3+, and Cr3+ as revealed by high association constants 12.27 × 103 M-1, 13.62
× 103 M-1, 33.56 × 103 M-1, respectively. Micro molar level response with very little interference
from other biologically competitive metal ions certainly supports its effectiveness as a
chemosensor. In vitro fluorescence imaging by fluorescence microscope helps to understand
the chemical basis of Al3+, and Cr3+ activity in the biological system whereas paper strip ex-
periment ensures the usability of sensor L as convenient practical a solid probe for onsite anal-
ysis of metal Al3+ and Cr3+ ions. Therefore, we believe that this type of sensor will pave the
way for the strategic architecture of a single sensor for multiple analytes detection.
Author Statement
The Authors declare that there is no conflict of interest.
Conflicts of interest
There is no conflict of interest to declare.
Acknowledgments
S.M. is thankful to NIT, Durgapur for providing the research facilities. S. Dey thanks
University Grants Commission, New Delhi, India, for financial support by sanctioning a Minor
Research Project (Sanction Letter No. PSW-155/14-15(ERO) ID No. WV6-027, S. No. 223832
dated 03-02-2015). We thank Dr. Asim Kr. Bera, Principal, Mahisadal Raj College, and Dr.
Abdul Motin, Principal, Tamralipta Mahavidyalaya for providing laboratory and instrumental
facilities.
27

28
References:
[1]
[2]
[3]
L. M. Gaetke, C.K. Chow, Copper toxicity, oxidative stress, and antioxidant nutrients,
Toxicology. 189 (2003) 147–163.
M. Otto, LookingToward Basic Science for Potential Drug DiscoveryTargets Against
Community-Associated MRSA, Med Res Rev. 30 (2010) 1–22.
A. K. Bhanja, S. Mishra, K. Naskar, S. Maity, K. Das Saha, C. Sinha, Specific
recognition of Cr3+ under physiological conditions by allyl substituted appendage
rhodamine and its cell-imaging studies, Dalt. Trans. 46 (2017) 16516–16524.
[4]
O. Sunnapu, N.G. Kotla, B. Maddiboyina, G.S. Asthana, J. Shanmugapriya, K. Sekar,
S. Singaravadivel, G. Sivaraman, Rhodamine based effective chemosensor for
Chromium(III) and their application in live-cell imaging, Sensors Actuators, B Chem.
246 (2017) 761–768.
[5]
[6]
[7]
N.E.W. Alstad, B.M. Kjelsberg, L.A. Vøllestad, E. Lydersen, A.B.S. Poléo, The
significance of water ionic strength on aluminium toxicity in brown trout (Salmo trutta
L.), Environ. Pollut. 133 (2005) 333–342.
T. Han, X. Feng, B. Tong, J. Shi, L. Chen, J. Zhi, Y. Dong, A novel “turn-on” fluorescent
chemosensor for the selective detection of Al3+ based on aggregation-induced emission,
Chem. Commun. 48 (2012) 416–418.
A. Sahana, A. Banerjee, S. Lohar, A. Banik, S. K. Mukhopadhyay, D. A. Safin, M. G.
Babashkina, M. Bolte, Y. Garcia, D. Das, FRET-based tri-color emissive rhodamine-
pyrene conjugate as an Al3+ selective colorimetric and fluorescence sensor for living cell
imaging, Dalt. Trans. 42 (2013) 13311–13314.
[8]
[9]
J. R. Walton, Aluminum in hippocampal neurons from humans with Alzheimer’s
disease, Neurotoxicology. 27 (2006) 385–394.
Y. Yang, Y.-L. Wei, X.-M. Li, R.-R. Zhao, R. Tan, J.-F. Zhang, Y. Zhou, X.W. Lv, A
Rhodamine-based fluorescent sensor for chromium ions and its application in
bioimaging, Chinese Chem. Lett. 28 (2017) 1258–1261.
[10] A. Ghorai, J. Mondal, S. Chowdhury, G.K. Patra, Solvent-dependent fluorescent-
colorimetric probe for dual monitoring of Al3+ and Cu2+ in aqueous solution: An
28

29
application to bio-imaging, Dalt. Trans. 45 (2016) 11540–11553.
[11] X. Yu, H. Qu, Q. Hu, K. Hou, Y. Yan, D. Wu, P. Zhang, J. Li, A rhodamine-based sensor
for chromogenic detection of Cu2+ and fluorescent detection of Fe3+, Wuhan Univ. J.
Nat. Sci. 19 (2014) 129–136.
[12] P. Kaur, D. Sareen, The synthesis and development of a dual-analyte colorimetric
sensor: Simultaneous estimation of Hg2+ and Fe3+, Dye. Pigment. 88 (2011) 296–300.
[13] N. Singh, N. Kaur, C. Ni Choitir, J.F. Callan, A dual detecting polymeric sensor:
chromogenic naked-eye detection of silver and ratiometric fluorescent detection of
manganese, Tetrahedron Lett. 50 (2009) 4201–4204.
[14] D. Y. Lee, N. Singh, D.O. Jang, A benzimidazole-based single molecular multianalyte
fluorescent probe for the simultaneous analysis of Cu2+ and Fe3+, Tetrahedron Lett. 51
(2010) 1103–1106.
[15] Z. Q. Hu, M. Du, L.F. Zhang, F.Y. Guo, M.D. Liu, M. Li, A novel colorimetric and
fluorescent chemosensor for cyanide ion in aqueous media based on a rhodamine
derivative in the presence of Fe3+ ion, Sensors Actuators, B Chem. 192 (2014) 439–443.
[16] A. Mohammadi, Z. Dehghan, M. Rassa, N. Chaibakhsh, Colorimetric probes based on
bioactive organic dyes for selective sensing of cyanide and fluoride ions, Sensors
Actuators, B Chem. 230 (2016) 388–397.
[17] J. Griffiths, II. Photochemistry of azobenzene and its derivatives, Chem. Soc. Rev. 1
(1972) 481–493.
[18] A. Bafana, S.S. Devi, T. Chakrabarti, Azo dyes: Past, present, and the future, Environ.
Rev. 19 (2011) 350–370.
[19] S. Paul, A.Manna and S.Goswami, A differentially selective molecular probe for
detection of trivalent ions (Al3+, Cr3+, and Fe3+) upon single excitation in a mixed
aqueous medium, Dalt. Trans. 26 (2015) 11805–11810.
[20] R. G. Parr and W. Yang, Density Functional Theory of Atoms and Molecules, Oxford
Univ. Press. (1989).
[21] V. Barone, M. Cossi, Conductor Solvent Model, J. Phys. Chem. A. 102 (1998) 1995–
2001.
29