From one to three, modifications of sensing behavior with solvent system: DFT calculations and real-life application in detection of multianalytes (Cu2+, Ni2+ and Co2+) based on a colorimetric Schiff base probe

Article abstract of DOI:10.1016/j.molstruc.2021.130453

A rhodamine-based colorimetric sensor, RBOV has been synthesized and modified for selective detection of Cu²⁺, Ni²⁺ and Co²⁺ ions simultaneously in aqueous medium. The difference in color change upon binding with these three cations makes it an interesting multianalyte sensor. Furthermore, the detection limits of RBOV for Cu²⁺, Ni²⁺ and Co²⁺ are down to 4.0 × 10^?2 μM, 3.2 × 10^?1 μM and 4.8 × 10^?1 μM respectively. The possible sensing mechanism analyzed from Benesi-Hildebrand plot, Job's plot, FTIR and DFT studies showed a 1:1 stoichiometry of sensor-metal complex for all three analytes. Practical application of sensor was conducted in real time using test strips and MTT assay was conducted to determine its cytotoxicity.

Full text of DOI:10.1016/j.molstruc.2021.130453

Journal of Molecular Structure 1238 (2021) 130453
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
From one to three, modifications of sensing behavior with solvent
system: DFT calculations and real-life application in detection of
2
+
2+
2+
multianalytes (Cu , Ni and Co ) based on a colorimetric Schiff
base probe
a
b
b
b
Nur Amira Solehah Pungut , Mok Piew Heng , Hazwani Mat Saad , Kae Shin Sim ,
a
a,∗
Vannajan Sanghiran Lee , Kong Wai Tan
a
Department of Chemistry, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia
Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A rhodamine-based colorimetric sensor, RBOV has been synthesized and modified for selective detection
Received 24 November 2020
Revised 26 March 2021
Accepted 7 April 2021
Available online 13 April 2021
2+
2+
2+
of Cu , Ni
and Co
ions simultaneously in aqueous medium. The difference in color change upon
binding with these three cations makes it an interesting multianalyte sensor. Furthermore, the detection
limits of RBOV for Cu2 , Ni and Co are down to 4.0 × 10 μM, 3.2 × 10 μM and 4.8 × 10 μM re-
spectively. The possible sensing mechanism analyzed from Benesi-Hildebrand plot, Job’s plot, FTIR and
DFT studies showed a 1:1 stoichiometry of sensor-metal complex for all three analytes. Practical applica-
tion of sensor was conducted in real time using test strips and MTT assay was conducted to determine
its cytotoxicity.
+
2+
2+
−2
−1
−1
Keywords:
Rhodamine B
Chemosensors
Colorimetric
Test strips
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
emission spectrometry (ICP-AES), inductively coupled plasma mass
spectrometry (ICP-MS), and atomic absorption spectroscopy (AAS)
were used. Although the use of said instruments serves its pur-
pose, compared to detection via chemosensors, it is rather expen-
sive and the overall process is time consuming [17–19]. On the
other hand, colorimetric chemosensors ability in providing ‘’naked
eye’’ detection has been of great importance as this enables rapid
qualitative and quantitative information without any need of ex-
pensive instrumentations [20,21].
Copper, being one of the essential trace element plays a pro-
found role in the physiological and biochemical processes such as
its important role in antioxidant defense as copper-zinc superoxide
dismutase [1,2] . On the other hand, nickel plays a crucial role in
biological functions such as aerobic and anaerobic respiration, and
biosynthesis of enzymes [3–5] . Although cobalt is not as abundant
as copper and nickel, it is still one of the essential elements found
in Vitamin B12 and plays a vital part in the metabolism of iron
and the synthesis of hemoglobin [6,7]. Even though the presence
of these metal ions is beneficial in trace amount, a surplus of these
ions will be detrimental to the human health. Such adverse effects
may include, Alzheimer’s disease [8], Parkinson’s [9] and Wilson
disease [10] for copper ion, respiratory system [11] and systemic
contact dermatitis [12] for nickel ion, and cardiac related diseases,
thyroid enlargement, polycythemia, vasodilation and flushing for
cobalt ions [13–16].
Ever since the first rhodamine-based chemosensor developed
by Czarnik’s group back in 1997 [22], rhodamine dyes have been
widely used in the development of chemosensors. This is due to its
exceptional spectroscopic attributes such as high absorption coeffi-
cient, absorption and emission at longer wavelength and excellent
quantum yields [23,24]. Generally, in the closed spirolactam ring
form, rhodamine derivatives are colorless and non-fluorescent. A
rapid color change would be observed upon opening of the spiro-
lactam ring, by the noticeable pink color and heightened fluores-
cent emission [25–27].
Before detection of metal ions was done by chemosensors,
conventional methods such as inductively coupled plasma atomic
In this work, a rhodamine B based chemosensor, RBOV has
been synthesized with slight modifications from previous pub-
lished methods [28,29]. The sensor is formed from the reaction of
rhodamine B hydrazide with 2–hydroxy-3-methoxybenzaldehyde
in a two-step reaction (Scheme 1). Previous work on this sensor
∗
Corresponding author.
E-mail address: kongwai@um.edu.my (K.W. Tan).
https://doi.org/10.1016/j.molstruc.2021.130453
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
Scheme. 1. Synthesis of RBOV Sensor.
has reported its ability in detecting single metal ion (Cu2 ) and
bioimaging in maize roots [30]. In the current work, we demon-
strated a simple and cost-effective strategy in bestowing a known
sensor with multiple cations recognition behavior through care-
ful optimization of different solvent system. DFT calculations were
performed to elucidate the sensing behavior and cell viability as-
say was carried out to determine the potential of the sensor for
cell staining. Furthermore, it is the first sensor known that can de-
+
formed on a Shimadzu UV-2600 spectrophotometer with cell path
length of 1 cm. All spectral assay was carried out in the range of
300–700 nm. The melting points was recorded using the Mel-Temp
II apparatus.
2
.2. Synthesis of RBH
Rhodamine B hydrazide, RBH was synthesized by following a
tect multiple analytes such as Cu2 , Ni , and Co simultaneously.
Previous works on detection of multiple ions [31–34] have been
limited to the detection of either one, or two of the metal ions
+
2+
2+
previous described method [22]. To a (0.50 g, 1.0 mmol) of rho-
damine B dissolved in 10 mL of ethanol, and hydrazine hydrate
(
1.0 mL) was added dropwise. The solution was then refluxed for
h until the color changes from purple to orange. After that, the
stated. Other than that, binding of sensor towards Ni2 and Co
+
2+
6
can be differentiated by carrying further test and the practicality of
this sensor in detecting all three metal ions in real time as on-site
assay kit was conducted.
convection of the solution to room temperature, it was then ex-
tracted with dilute hydrochloric acid (HCl, 1 M) and sodium hy-
droxide (NaOH, 1 M). The precipitate formed was then collected
and separated by percolation and was washed with 45 mL of dis-
tilled water. Finally, the precipitate is left to dry and was then re-
crystallize with ethanol in a closed conical flask for slow evapora-
tion. The RBH (yield: 85%, 0.85 mmol) obtained was then charac-
terized by using FTIR, 1H NMR and 13C NMR. 1H NMR (400 MHz,
2. Experimental
2
.1. Materials and instrumentation
DMSO–d , TMS, s = singlet; d= doublet; t = triplet; q = quadruplet;
6
Ethanol and DMSO were purchased from MERCK while 2–
m = multiplet), δ (ppm): 1.09 (t, 12H, NCH CH ); 3.31 (q, 8H,
2
3
hydroxy-3-methoxybenzaldehyde and rhodamine
B were pur-
NCH CH ); 4.22 (s, 2H, NH ); 6.34 (d, 2H, Xanthene-H); 6.38 (d,
2
3
2
chased from Alfa Aesar. The stock solution of RBOV (100 mM) was
prepared by dissolving the requisite amount of it in DMSO. Stock
solutions (100 mM) of various metal ions were procured by using
befitting amount of distilled water. While tris-sodium chloride (TN)
buffer was made by using befitting amount of corresponding salt
and acid.
2
H, Xanthene-H); 6.99 (d, 2H, Xanthene-H); 7.48 (m, 2H, Aromatic-
H); 7.78 (m, 1H, Aromatic-H). 3C NMR (400 MHz, DMSO–d6,
1
TMS), δ (ppm): 12.97 (NCH CH ); 44.21 (NCH CH ); 65.30 (C–N);
2
3
2
3
97.94, 105.95, 108.32, 122.69, 124.01, 128.21, 128.65, 130.11, 132.92,
1
48.64, 148.79, 152.40, 152.89, 153.56 (Aromatic-C); 165.85 (C=O).
IR spectra were recorded by using KBr pellets with Perkin-
Elmer Spectrum 400 RX-1 spectrometer. NMR were recorded in
deuterated DMSO on a JEOL ECX 400 MHz instrument. Elemental
analysis was carried out on a Perkin Elmer Series II 2400 CHNS ele-
mental analyzer. Jeol AccuTOF Mass Spectrometer was used to per-
form the mass spectrometry. UV–Vis spectral analyses were per-
2.3. Synthesis of RBOV
RBOV was synthesized by reacting RBH with 2–hydroxy-3-
methoxybenzaldehyde (also known as ortho-vanillin) in ethanol.
To a 100 mL flask, rhodamine B hydrazide (0.20 g, 0.4 mmol) was
2

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
dissolved in 25 mL ethanol, and ortho-vanillin (0.06 g, 0.4 mmol)
was added into the mixture and refluxed for 6 h. The reaction
mixture was then concentrated and dried at room temperature.
Red product (yield: 82%, 0.33 mmol) obtained was then recrys-
tallized with ethanol and placed in room temperature for slow
evaporation. Melting point (m.p.)= 176–178 °C 1H NMR (400 MHz,
DMSO–d , TMS, s = singlet; d= doublet; t = triplet; q = quadruplet;
6
Fig. 1. Colorimetric changes of RBOV (50 μM) upon addition of various metal ions
(25 μM) in DMSO/TN solution (2:1, v/v, pH 7.5. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this arti-
cle.)
m = multiplet), δ (ppm): 1.01 (t, 12H, NCH CH ); 3.24 (q, 8H,
2
3
NCH CH ); 3.67 (s, 3H, O–CH ); 6.28 (d, 1H, Xanthene-H); 6.30
2
3
3
(
d, 1H, Xanthene-H); 6.37 (q, 4H, Xanthene-H); 6.70 (t, 1H,
Xanthene-H); 6.83 (dd, 1H, Aromatic-H); 6.89 (dd, 1H, Aromatic-
H); 7.05 (d, 1H, Aromatic-H); 7.55 (m, 2H, Aromatic-H); 7.87
dd, 1H, Aromatic-H); 10.18 (s, 1H, C–OH). 13C NMR (400 MHz,
(
DMSO–d , TMS), δ (ppm): 12.85 (NCH CH ); 49.09 (NCH CH );
6
2
3
2
3
5
6.59 (O–CH ); 66.06 (C–N); 97.77, 105.21, 108.64, 114.36, 119.51,
3
1
21.19, 123.57, 124.35, 128.19, 129.43, 147.43, 148.33, 149.07, 151.52,
53.23 (Aromatic-C); 164.13 (C=O). CHNS elemental analysis (%)
for C N O (RBOV containing 1 water molecule): Calculated:
1
H
3
6
40
4
5
C = 71.03; H = 6.62; N = 9.20; Found C = 71.23 (± 0.2); H = 6.65
(
± 0.03); N = 9.41 (± 0.21); m/z = 608.30. HRESIMS m/z calculated
for C
H
N O [M + H]+ = 591.2978; found= 591.2971.
3
6
39
4
4
2
.4. FT-IR analysis of metal complexes
The sensor-metal complexes, RBOV-Cu2 /Ni /Co
+
2+
2+
were pre-
pared by reacting RBOV (0.10 g, 0.1 mmol) with the respected metal
salts (0.2 mmol) in ethanol (20 mL) at room temperature. Slow
evaporation of the reaction mixtures yielded solids suitable for
FTIR characterization.
Fig. 2. Absorption changes of RBOV (50 μM) upon addition of various metal ions
(
including the free sensor, Fs) (25 μM) in DMSO/TN solution (2:1, v/v, pH 7.5).
2
.5. Cell culture and MTT cytotoxicity assay
The human colorectal adenocarcinoma (HT-29) and normal
colon fibroblast (CCD-18Co) were purchased from American Type
Culture Collection (ATCC, USA). The cells were cultured in McCoy’s
5
A medium (HT-29), and T75 medium (CCD-18Co), supplemented
with 10% fetal bovine serum and incubated at 37°C in a humidified
atmosphere containing 5% CO2.
The MTT assay was performed as described by Mosmann with
3
2+
2+
and RBOV-Co
modifications [35]. Briefly, the HT-29 (2 × 10 cells/well) and CCD-
Fig. 3. Color changes upon addition of H2O2 towards RBOV-Ni
3
complexes. (For interpretation of the references to color in the text, the reader is
referred to the web version of this article.)
1
8Co (10 × 10 cells/well) cells were seeded into a 96-well mi-
croplate and incubated overnight prior the treatment with various
concentrations of RBOV probe. After 72 h of incubation, 20 μL of
MTT solution (5 mg/mL) was added into each well and incubated
for another 3 h. The medium was then removed, and the formazan
crystals were dissolved in 200 μL of DMSO. The absorbance of
each well was measured using Tecan M200 Infinite Pro-Microplate
Reader at 570 nm with 650 nm as reference wavelength. Cisplatin
was used as positive control in this study. The data was expressed
as mean ± standard deviation of triplicate experiments.
Since RBOV shows the same colorimetric changes upon binding
2+
2+
to Ni
and Co
ions, oxidizing agent was used in order to dif-
ferentiate the binding of sensor towards these metal ions. As de-
2+
picted in Fig. 3, only RBOV-Co
complex changes from yellow to
2+
pink, whereas RBOV- Ni complex remains unchanged upon ad-
dition of hydrogen peroxide, which correlates to the properties of
2+
the metal ion itself, as Ni is known for its resistant towards ox-
idation. The change in color for RBOV-Co2 complex suggests that
+
3. Results and discussion
2+
3+
Co has been oxidized to Co .
3
.1. Colorimetric detection of metal ions by RBOV
3
.2. Effects of pH on detection of metal ions
In order to elucidate the effectiveness of RBOV at various pH,
This study was performed with 11 different metal ions compris-
+
3+
2+
2+
2+
2+
2+
+
2+
2+
ing, Ag , Al , Cd , Cu , Co , Cr , Mn , Na , Ni , Pb and
Zn2 in DMSO/TN solution (2:1, v/v, pH 7.5). After the addition of
+
the response rate was studied in DMSO/TN solution with and with-
out presence of metal ions. As shown in Fig. 4, the free RBOV ab-
sorbs at pH 2 in resultant to the opening of spirolactam ring under
various metal ions, Cu2 , Ni and Co showed obvious changes in
absorbance which suggests the formation of delocalized xanthene
moiety of rhodamine and the opening of spirolactam ring. This
process could be observed in Fig. 1 with the obvious noticeable
+
2+
2+
acidic condition. In the presence of Cu2 ion, significant absorption
+
enhancement is observed at pH 2–7. Even so, a change in absorp-
appearance of pink color for Cu2 and yellow for Ni and Co . In
+
2+
2+
tion in the presence of Ni and Co occurs at pH 7–10. From the
results obtained, an optimal absorption response of RBOV towards
the target metal ions could be produced by conducting the assays
under physiological pH condition, thus pH 7.5 was chosen.
2+
2+
particular, the addition of Cu2 exhibit the highest change in ab-
sorbance centered at 560 nm, followed by Ni2 and Co at 425 nm
+
+
2+
as depicted in Fig. 2.
3

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
2
+
Fig. 4. Absorption changes of (a) RBOV, RBOV + Cu
(λ= 560 nm), and (b)
RBOV + Ni +, RBOV + Co2+ (λ= 425 nm) over a range of pH (2–10) in DMSO/TN so-
2
lution (2:1, v/v).
Fig. 5. Absorbance changes of RBOV (50 μM) upon increasing concentration of Cu2
+
at 560 nm in DMSO/TN solution (2:1, v/v, pH 7.5). Inset: Changes of absorbance in-
tensity with incremental addition of metal ions.
3
.3. Limit of detection
Next, the sensitivity of RBOV was determined by conducting
titration experiments where concentrations of Cu2 , Ni and Co
2+
+
2+
were varied from 0 to 25 μM, with interval of 2.5 μM at neutral
pH. A graph of relative absorbance at 560 nm for Cu2+ ions was
Fig. 6. Relative absorbance of RBOV (50 μM) towards a) Cu2 at λ= 560 nm, b) Ni
+
2+
and Co2+ at λ= 425 nm, (50 μM), in the presence of other metal ions (50 μM) in
DMSO/TN solution (2:1, v/v, pH 7.5).
plotted as a function of concentration as depicted in Fig. 5. Graphs
of relative absorbance for Ni2 and Co
+
2+
ions could be observed
as in Figure S1 and S2 respectively. The detection limit of RBOV
Cu2 , Ni
2+
50 μM) in the presence of other metal ions Ag , Al , Cd , Cr ,
+
2+
and Co , competitive selectivity of the sensor
2+
was calculated from the equation, DL= 3S /m, where S is the
d
d
+
3+
2+
(
standard deviation of blank measurement and m is the slope be-
tween absorbance intensity and the metal concentration (Inset of
Fig. 5). From the formula, the limit of detection (LOD) values of
Mn2 , Na , Pb
+
+
2+
and Zn (50 μM) were evaluated (Fig. 6). From
+
2+
Fig. 6, there’s only slight interference on the absorbance of Cu2
Ni2 and Co , in the presence of other metal ions. For the de-
tection of Co2 , an increment in absorbance was observed upon
presence of Ni2 . This result was expected as RBOV sensor detects
,
+
2+
+
−
2
−1
RBOV have been determined to be 4.0 × 10 μM, 3.2 × 10 μM
−1
2+
2+
2+
and 4.8 × 10 μM for Cu , Ni and Co ions respectively. These
+
values are much lower than the recommended water quality stan-
Ni2 at a higher absorbance than Co at similar wavelength.
+
2+
dard for Cu2 (3.1 × 10 μM), Ni (1.2 × 10 μM) and Co (average
+
1
2+
1
2+
1
of 3.7 × 10 μM), in drinking water by WHO [36,37] . Thus, indi-
3
.5. Binding stoichiometry
cating that the probe is highly sensitive and selective in detecting
these metal ions.
In order to elucidate the stoichiometry of RBOV and the metal
ions (Cu2 , Ni , and Co ), Job’s plot analysis was done. In this
analysis, the mole fraction of target analyte was varied from 0 to 1
and the total concentration of sensor and target analyte was fixed
at 50 μM (Fig. 7). In all three cases, the curve maximum corre-
sponded to a ∼0.6 mol fraction, indicating a 1:1 complex between
RBOV and the three metal ions.
+
2+
2+
3
.4. Selectivity of RBOV amid interfering cations
Other than sensitivity, selectivity is also one of the important
properties for a sensor detecting multiple metal ions. To further
investigate the selectivity of RBOV in detecting
4

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
Fig. 8. Shifting of peaks from FTIR spectrum of RBOV upon binding to the metal
ions.
3
.6. FT-IR analysis
To confirm the binding mechanism, IR (Fig. 8) spectra of RBOV
+
and RBOV-M complex was analyzed by using KBr pellets, and the
change in its characteristic bands in the double bond region was
observed. The two bands are known as the ketone carbonyl and
amide carbonyl. The ketone carbonyl and amide carbonyl bands of
RBOV were found at 1717 cm 1 and 1610 cm respectively. Upon
− −1
Fig. 7. Job’s plots of RBOV and M2 ([M ] + [RBOV] = 50 μM) at λ= 560 nm for a)
+
2+
2+
Cu2 and at λ= 425 nm for b) Ni2+and M ([M ] + [RBOV] = 25 μM) for c) Co in
+
2+
2+
2+
binding to Cu , the shifting of peak occurs and was observed at
DMSO/TN (2:1, v/v, pH 7.5) solution.
−1
1
585 cm (Fig. 8(b)). Similar observations were recorded from the
IR spectra of RBOV-Ni2 and RBOV-Co
+
2+
(Fig. 8(c) and (d)). This
shows that both the ketone and amide carbonyl group of RBOV are
involved in the coordination with metal cations.
As a 1:1 binding stoichiometry was obtained from Job’s plot for
the binding of RBOV to the metal ions, the association constant,
Ka was determined using the Benesi-Hildebrand equation [38] as
follow
3
.7. Computational study
The density functional theory (DFT) calculations were per-
formed by Gaussian 09 Rev C.01, in order to further explain the
interactions between RBOV sensor and cations. The structures of
1
1
1
+
+
2+
2+ 2+
and Co ) were op-
=
+
(1)
RBOV and RBOV-M (where M = Cu , Ni
A − A0
Ka(Amax − A0)[M+]
Amax − A0
timized by using B3LYP [39–41] level together with the LANL2DZ
basis set. Then the frequencies calculation was performed to con-
firm the true minimum. The binding energy between the RBOV and
each metal ion in vacuo is defined by the following formula:
A and A0 is the absorbance of RBOV solution in the presence
+
and absence of M respectively; Amax is the saturated absorbance
+
+
of RBOV in the presence of excess amount of M ; [M ] is the con-
ꢀ
(binding energy) = E(metal − binding RBOV ) − E(RBOV )
E(free metal ion)
We employed two basis sets using the density function the-
centration of metal ions added.
+
+
By plotting a graph of 1/(A-A ) versus 1/[M ] (Figure S3), a
0
linear regression equation was obtained for all three metal ions.
Thus, the value of Ka obtained for RBOV-metal ions complex were,
ory; B3LYP/LANL2DZ by means of Rayleigh–Schrödinger perturba-
tion theory (RS-PT) and second-order Møller–Plesset perturbation,
MP2/LANL2DZ which improves on the Hartree–Fock method by
4
−1
4
−1, and 8.6 × 104
−1
2+
2+
1
.8 × 10
M
, 4.4 × 10
M
M
for of Cu , Ni ,
2
+
and Co respectively.
5

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
Fig. 9. The optimized structures of RBOV-metal and molecular orbitals calculated at B3LYP/ LANL2DZ level for (a) RBOV, (b) RBOV-Ni2 , (c) RBOV-Cu and d) RBOV-Co
2+
.
+
2+
The binding energy was calculated using B3LYP/ LANL2DZ and MP2// LANL2DZ basis sets.
all over the imine moiety for RBOV, RBOV-Ni2 , RBOV-Cu , and
+
2+
adding electron correlation effects in order to get more accurate
complexes metal binding energy. From previous reports, DFT tech-
nique with B3LYP/LANL2DZ has shown promising results on the
predicted complex structures in various applications [42,43].
As depicted in Fig. 9, the electron density of in the HOMO
spreads over the xanthene moiety, whereas the LUMO spreads
RBOV-Co2 . The HOMO and LUMO energy gaps between RBOV and
+
RBOV-(Ni2 , -Cu
+
2+
and Co ) was calculated to be 3.64 eV and
2+
2.82 eV, 2.59 eV and 2.39 eV. All three metal complexes show a
binding energy which is at least 20% lesser than that of RBOV
showing an increase in stabilization upon binding. This further
6

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
Fig. 10. Changes of absorption intensity in DMSO/TN (2:1, v/v, pH 7.5) solution at
2
+
2+
λ= 560 nm of i: RBOV (25 μM) with Cu (25 μM); ii: RBOV with Cu upon addi-
2
+
tion of EDTA (50 μM); and iii: absorbance upon re-addition of Cu (50 μM). Inset:
Images of color change during addition of EDTA and re-addition of Cu2 . (For in-
terpretation of the references to color in the text, the reader is referred to the web
version of this article.)
+
supports the binding mode of metal ions with RBOV sensor as sug-
gested by Job’s plot and FT-IR analysis.
The optimized structures of 1:1 (RBOV: metal) ratio and the
bond lengths between each metal ion and its ligands (RBOV) pro-
vide a clear picture about the three-dimensional arrangement of
different atoms in the complexes as illustrated in Fig. 9. The pre-
dicted structures are similar to previous investigation by Chaud-
hary and Mishra, 2017 for Schiff base metal complexes [44] where
the electron density region is around two O-atoms and one N-atom
that were bonded to the metal ion. This is the region of stabil-
ity for coordinated metal ions and RBOV. Similar study and com-
putational data of the complexes are in good support of the pro-
posed structures. The binding strength was revealed using the cal-
culated binding energy. Among the three RBOV metal complexes,
the correlated results were found using the electron correlation
technique, MP2/LANL2DZ, where the binding strength was ranked
Fig. 12. Cytotoxic activity of RBOV against a) CCD-18Co and b) HT-29 cell lines. Cis-
platin was used as positive control. Cells were treated with different concentrations
of RBOV at 37 °C for 72 h. Data is expressed as mean ± SD (n = 3). .
from RBOV-Ni2 > RBOV-Cu > RBOV-Co with the value energy
+
2+
2+
of −733.35, −687.58, and −671.82 kcal/mol.
(
2:1, v/v, pH 7.5) solution. As seen in Fig. 10, it can be clearly
observed that upon addition of EDTA into the system containing
3
.8. Reversibility
RBOV-Cu2 complex, a change in color, from pink to colorless oc-
+
curs. Whereas, an increment in absorbance was observed upon re-
+
2+
In developing a chemosensor, reversibility and regeneration of
addition of Cu2 . The same changes were observed for Ni
and
Co2 ions (Figure S4 and S5). From the outcome, it could be in-
+
a sensor from sensor-metal complex is an important aspect in
terms of practical application. The reversible nature of RBOV was
investigated by doing an EDTA addition experiment in DMSO/TN
ferred that complexation of RBOV towards Cu2 , Ni , and Co
+
2+
2+
are reversible.
Fig. 11. Colorimetric changes of test strips upon immersing in increasing concentrations of metal ions. (For interpretation of the references to color in the text, the reader is
referred to the web version of this article.)
7

N.A.S. Pungut, M.P. Heng, H.M. Saad et al.
Journal of Molecular Structure 1238 (2021) 130453
3
.9. On-site assay study
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A Long-wavelength fluorescent chemod-
Nur Amira Solehah Pungut: Writing- original draft, Validation,
Investigation, Visualization, Data curation, Methodology, Conceptu-
alization. Mok Piew Heng: Validation, Investigation, Methodology,
Conceptualization, Hazwani Mat Saad: Methodology, Investigation,
Validation, Kae Shin Sim: Writing - review & editing, Vannajan
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