A turn on fluorescent sensor for detecting Al3+ and colorimetric detection for Cu2+: Synthesis, cytotoxicity and on-site assay kit

Article abstract of DOI:10.1016/j.jphotochem.2021.113290

A new rhodamine-fluorene based sensor, RFC has been synthesized for selective detection of Al³⁺ and Cu²⁺ with detection limit as low as 0.12 μM and 1.14 μM respectively. The “off-on’’ colorimetric changes of RFC upon binding with the metal ions was differentiated by UV-light. Furthermore, the possible sensing mechanism was proposed from FTIR and ¹H NMR spectrum, while the binding stoichiometry was determined from Benesi-Hildebrand and Job's plot. From these studies, a 1:1 stoichiometry of sensor-metal complex for both RFC-Al³⁺ and RFC-Cu²⁺ was recorded. On-site virtual detection of these metal ions by RFC was done by using filter paper as test strips. MTT assay indicated that RFC have no significant cytotoxicity to the cultured human colorectal adenocarcinoma (HT-29) and normal (CCD-18Co) cells.

Full text of DOI:10.1016/j.jphotochem.2021.113290

Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
3
+
A turn on fluorescent sensor for detecting Al and colorimetric detection
2
+
for Cu : Synthesis, cytotoxicity and on-site assay kit
a
b
b
a,
Nur Amira Solehah Pungut , Hazwani Mat Saad , Kae Shin Sim , Kong Wai Tan *
a
Department of Chemistry, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia
b
Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia
A R T I C L E I N F O
A B S T R A C T
A new rhodamine-fluorene based sensor, RFC has been synthesized for selective detection of Al3 and Cu with
+
2+
Keywords:
Rhodamine B
Chemosensors
MTT
detection limit as low as 0.12 M and 1.14
μ
μM respectively. The “off-on’’ colorimetric changes of RFC upon
binding with the metal ions was differentiated by UV-light. Furthermore, the possible sensing mechanism was
proposed from FTIR and 1H NMR spectrum, while the binding stoichiometry was determined from Benesi-
Test strips
3
+
Hildebrand and Job’s plot. From these studies, a 1:1 stoichiometry of sensor-metal complex for both RFC-Al
and RFC-Cu2 was recorded. On-site virtual detection of these metal ions by RFC was done by using filter paper
as test strips. MTT assay indicated that RFC have no significant cytotoxicity to the cultured human colorectal
adenocarcinoma (HT-29) and normal (CCD-18Co) cells.
+
1
. Introduction
Development of fluorogenic and chromogenic chemosensors have
abundant element in the Earth’s crust, it’s not surprising that aluminium
is easily exposed to the general population on the day-to-day basis.
Having being used extensively such as in pharmaceuticals [31], auto-
motive [32], textile industry [33], food additive [34] and treatment of
water [35], accumulation of excessive amount of this metal would have
adverse effects to the environment and human health [36,37]. Deposi-
tion of excess aluminium in its ionic state would lead to acidification of
soil [38] and known to cause neurodegenerative disease such as Par-
kinson’s disease [39] and kidney damage [40].
Generally, fluorene and its derivatives are known to have high
quantum yield making it highly photoluminescence [41,42]. Because of
this, it is widely utilized in LEDs [43], two photon absorption [44] and as
fluorescent sensors [45,46]. Furthermore, numerous reports have shown
a greater detection limit with fluorene based chemosensors [47–50].
Therefore, we have conjugated rhodamine B hydrazide with a fluorene
derivative (fluorene-2-carboxaldehyde) in this study.
been done extensively over the last decade [1–3]. Among the sensors
developed, rhodamines-based chemosensors have been preferred ever
since the work done back in 1997 [4] owing to its excellent photo-
physical properties [5]. With the “off-on’’ switch mechanism of these
chemosensors, rapid detection via naked-eyes could provide both
qualitative and quantitative information regarding the analytes detected
[
6–8] which could be utilized in developing an on-site assay kit [9,10].
Since there is an increasing use of heavy metal ions in the industry,
environmental pollution and health hazards are inevitable [11,12].
Although detection of metal ions by conventional methods exists, they
are less attractive as compared to chemosensors which are more
portable, cost effective and time-efficient [13–15].
In recent years, multitudes of rhodamine-based chemosensors have
3
+
been developed for detecting multiple metal ions such as Al [16,17],
Herein, the synthesis and characterization of a new rhodamine-
fluorene based chemosensor, RFC has been described. Naked eye
2
+
2+
3+
3+
Cu [18,19], Hg [20,21], Fe [22,23] and Cr [24,25]. Among
these metal ions, copper is one of the essential trace elements that plays
a major role in sustaining all forms of life and is directly involved in
physiological processes of the human body such as copper homeostasis
detection of Al3 and Cu by “off-on’’ switch mechanism enables rapid
+
2+
detection of these ions by RFC. Furthermore, enhancement of fluores-
cence emission for Al3 made the detection of the two ions easily
distinguishable. Previously, we have reported the synthesis of a fluo-
rescent chemosensor (MEK), obtained from the conjugation of rhoda-
mine B hydrazide and methyl ethyl ketone [51]. Although the sensor
+
[
26,27]. However, surplus amount of copper may lead to ecological
pollution and poses health hazards such as, Alzheimer’s [28], Menkes
29], and Wilson diseases [30]. On the other hand, being the third most
[
*
Corresponding author.
E-mail address: kongwai@um.edu.my (K.W. Tan).
https://doi.org/10.1016/j.jphotochem.2021.113290
Received 25 February 2021; Received in revised form 28 March 2021; Accepted 2 April 2021
Available online 14 April 2021
1
010-6030/© 2021 Elsevier B.V. All rights reserved.

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
M (Al3 ) and 5.10
+
μ
M (Sn ),
2+
2064878.
displayed great detection limit of 8.96
μ
detection of Al3 and Cu by RFC sensor with fluorene as its conjugate,
+
2+
showed greater detection limit of 0.12
μM and 1.14
μM respectively.
2.4. X-ray crystallography
Besides, addition of excess EDTA into RFC-metal ion complex shows
great reusability of RFC sensor, which is important for practical appli-
cation. On-site virtual detection of these ions seems promising as test
strips have been successfully prepared in aqueous media. The potenti-
ality of RFC in detecting these metal ions in living cells were also studied
by conducting MTT assay.
The RFC sensor was crystallized from dimethylformamide (DMF).
The crystal data of RFC was analyzed using Oxford Rigaku SuperNova
Dual diffractometer equipped with a Mo-Kα X-ray source (λ = 0.71073
Å) with Atlas detector to generate the unit cell parameter and intensity
data. Cell refinement, data acquisition and reduction were carried out
with CrysAlis Pro software [52]. The structure solution and refinements
were done by using SHELXL97 [53]. Crystal visualization was done by
using ORTEP3v2 [54] while the crystallographic information was edited
and formatted by using publCIF [55].
2
. Experimental
2
.1. Materials and instrumentation
Acetonitrile and chloroform were purchased from MERCK while
2.5. Stock solution preparation for spectral detection
fluorene-2-carboxaldehyde and Rhodamine B were purchased from Alfa
Aesar. The metal ions stock solution was prepared from their chloride,
A stock solution of RFC (100 mM) was prepared in chloroform. All
+ 3+ 2+ 2+ 2+ 2+ 3+ 2+ +
nitrate and sulphate salt. NMR were recorded in deuterated CDCl
3
on a
the metal cations (Ag , Al , Ca , Cd , Co , Cu , La , Mn , Na ,
2
+
2+
2+
2+
JEOL ECX400 MHz instrument. FT-IR spectra were recorded on a Perkin-
Elmer Spectrum RX-1 spectrometer using the ATR method. Absorption
spectra were measured on a Shimadzu UV-2600 series spectrophotom-
eter. Cary Eclipse fluorescence spectrophotometer was utilized for
fluorescence measurements.
Ni , Pb , Sn and Zn ) of concentration 100 mM was prepared in
distilled water. Detection of metal ions solutions are freshly prepared for
every spectroscopic measurement.
2.6. UV–vis and fluorescence spectral studies
2
.2. Synthesis of RBH
All experiments were carried out in MeCN/H
2
O (9:1, v/v) at pH 7.5
O (9:1, v/v)
(
0.1 mM Trisꢀ HCl buffer). The solvent system of MeCN/H
2
Rhodamine B hydrazide, RBH was synthesized by following a pre-
was chosen as it is the optimum condition at which the RFC sensor shows
great solubility and sensitivity. To investigate the metal ion selectivity,
the test samples were prepared by placing 1 equiv. of the cation stock
vious described method [4]. To a (0.5 g, 1.0 mmol) of rhodamine B
dissolved in 10 mL of ethanol, an excessive hydrazine hydrate (1.0 mL)
was added dropwise. The solution was then refluxed for 6 h until the
colour changes from purple to orange. After that, the convection of the
solution to room temperature, it was then extracted with hydrochloric
acid (HCl) and sodium hydroxide (NaOH). The precipitate formed was
then collected and separated by percolation and was washed with 45 mL
of distilled water. Finally, the precipitate is left to dry and was then
recrystallize with ethanol in a closed conical flask for slow evaporation.
solution in 3 mL of the RFC solution (50 M). In the fluorescence mea-
μ
surements (slit: 5.0 nm), excitation was provided at 562 nm and emis-
sion was recorded from 550 to 700 nm.
2.7. MTT cytotoxicity assay
The HT-29 (human colorectal adenocarcinoma) and CCD18-Co
(normal colon fibroblast) cell lines from American Type Culture
Collection (ATCC, USA) were used in current study. The MTT assay was
performed as described by Heng et al. [56], with cisplatin as positive
control. The data was expressed as mean ± standard deviation of trip-
licate experiments.
1
The RBH obtained was then characterized by using FTIR, H NMR and
1
3
1
C NMR. Yield = 85 %. H NMR (400 MHz, CDCl
3
-d, s = singlet; d =
doublet; t = triplet; q = quadruplet; m = multiplet), δ (ppm): 1.15 (t,
1
2
7
2H, NCH
2 3 2 3 2
CH ); 3.32 (q, 8H, NCH CH ); 3.60 (s, 2H, NH ); 6.28 (dd,
H, Xanthene-H); 6.41 (d, 2H, Xanthene-H); 6.45 (d, 2H, Xanthene-H);
1
3
.09 (m, 1 H); 7.43 (m, 2 H); 7.92 (m, 1 H) (Aromatic-H). C NMR (400
-d), δ (ppm): 12.70 (NCH CH ); 44.45 (NCH CH ); 66.01
C-N); 98.03, 104.61, 108.10, 123.07, 123.91, 128.18, 130.11, 132.60,
MHz, CDCl
3
2
3
2
3
3. Results and discussion
(
1
48.96, 151.64, 153.94 (Aromatic-C); 166.24 (C = O).
3.1. Synthesis of RFC
2
.3. Synthesis of RFC
RFC sensor was formed through condensation reaction as illustrated
in Scheme 1 below. The RFC crystal was grown in DMF solution and was
characterized using X-ray crystallography. The molecular structure of
RFC can be seen in Fig. 1, where it clearly shows the unique spirolactam
ring with the lactam and xanthene moiety forming a vertical plane. The
crystal data and structure refinement parameters of RFC are shown in
Table S1.
RFC was synthesized by reacting RBH with fluorene-2-
carboxaldehyde in ethanol. To a 100 mL flask, rhodamine B hydrazide
0.20 g, 0.4 mmol) was dissolved in 15 mL ethanol, and fluorene-2-
(
carboxaldehyde (0.08 g, 0.4 mmol) was added into the mixture and
refluxed for 5 h. The reaction mixture was then concentrated and dried
at room temperature. Orange product obtained was then recrystallized
with DMF and placed in room temperature for slow evaporation. Yield =
3.2. Spectral studies of RFC using UV–vis and fluorescence spectroscopy
1
6
0 %. H NMR (400 MHz, CDCl
3
-d, s = singlet; d = doublet; t = triplet; q
=
quadruplet; m = multiplet), δ (ppm): 1.15 (t,12H, NCH
2
CH
3
); 2.90 (d,
In determining the efficiency of a sensor, the selectivity of RFC (50
+
3+
2+
2+
2+
2+
1
H); 3.32 (q, 8 H, NCH CH ); 3.83 (s, 1 H); 6.25 (dd, 2 H); 6.47 (d, 2 H),
2
3
μ
M) towards a series of metal ions (Ag , Al , Ca , Cd , Co , Cu ,
3
+ 2+ + 2+ 2+ 2+ 2+
6
7
8
.56 (d, 2 H)(Xanthene-H); 7.11 (dd, 1 H), 7.29 (m, 1 H), 7.48 (m, 4 H),
.65 (d, 1 H), 7.72 (d, 1 H), 7.81 (s, 1 H), 8.01 (dd, 1 H) (Aromatic-H);
La , Mn , Na , Ni , Pb , Sn and Zn ) was investigated using the
3
+
UV–vis and fluorescence spectroscopy. Upon addition of 1 eq. of Al
1
3
2+
.58 (s, 1 H, imine-H)
C NMR (400 MHz, CDCl
3
-d, TMS), δ
and Cu in MeCN/H
2
O (9:1, v/v, pH 7.5, Trisꢀ HCl buffer, 0.1 mM), an
(
ppm):12.72 (NCH
2
CH ); 36.83; 44.42 (NCH
3
2
CH ); 65.96 (C-N); 97.99;
3
obvious colour change from colourless to purple was observed, giving
rise to the potential of RFC as naked-eye detector for these metal ions. As
1
1
1
06.05; 108.19; 119.61; 120.26; 123.70; 125.15; 127.16; 128.31;
29.10; 133.42; 134.03; 141.24; 143.36; 144.00; 147.21; 149.05;
shown in Fig. 2, upon the coordination of RFC towards Al3 and Cu , a
peak is observed at 560 nm from the UV–vis spectra, and no peak was
observed for the other cations.
+
2+
52.23 153.09 (Aromatic-C); 165.15 (N-C = O). HRESIMS m/z calcu-
+
lated for C42
H
41
N
4
O
2
[M+H] = 632.7998; found = 633.3230. CCDC:
2

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
Scheme 1. Synthetic route of RFC.
Fig. 2. Absorption changes of RFC (50
μ
M) upon addition of various metal ions
(
50 M) in MeCN/H
μ
2
O (9:1, v/v, pH 7.5, Trisꢀ HCl buffer, 0.1 mM).
Fig. 1. ORTEP of RFC with atom numbering scheme (50 % thermal ellipsoid
probability). Hydrogen atoms were omitted for clarity.
3
.3. Effects of pH
As it is known that for rhodamine derivatives, spirolactam induced-
The fluorogenic behavior of RFC towards the various metal ions was
also conducted via the same reaction condition. In the absence of metal
ions, a weak band at 588 nm was observed, attributing to the closed
spirolactam ring [57] of RFC. When metal ions are added, a strong peak
ring opening may occur in acidic condition [58]. Therefore, the effect of
pH on the behavior of RFC in detecting Al3 and Cu was investigated
+
2+
in MeCN/H O (9:1, v/v) solution. Fig. S3 shows that pH value affects the
2
3
+
3+
2+
was observed for Al and a significantly low peak was observed for
absorbance of RFC towards Al and Cu . In the absence of metal ions,
2
+
Cu as depicted in Fig. 4. In Fig. 3b, it could be seen that there is a
strong orange fluorescence under the UV lamp which is only observable
RFC shows no absorbance from pH 2ꢀ 11. There are also no changes in
absorbance upon binding to Al3 and Cu from pH 8ꢀ 11. Both metal
ions show highest absorbance value at pH 7.5, thus the results indicate
that detection of RFC towards metal ions is optimum at pH 7.5.
+
2+
3
+
for Al , which is attributed to the opening of the spirolactam ring. Thus,
3
+
2+
enable an easy and simple method of differentiating Al and Cu upon
detection by RFC sensor.
3
.4. Titration experiment of RFC
In order to determine the sensitivity of RFC in detecting Al3 and
+
3

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
Fig. 3. a) Observed colour change of RFC upon addition of various cations b) Fluorescence change of RFC upon addition of various metal ions under UV-lamp.
LOD) of RFC towards these metal ions were determined. As shown in
(
insets of Fig. 5, linear fitting of the fluorescence and absorbance titration
profiles of Al3 and Cu were used in determining the LOD respec-
+
2+
tively. The LOD value of RFC was calculated based on the equation, DL =
d d
3S /m [59], where S is the standard deviation of blank measurement
and m is the slope between absorbance intensity and the metal con-
centration. The detection limit of Al3 and Cu was calculated as 0.12
M and 1.14 M respectively. These values are much lower than the
recommended water quality standard for Al (3.71
M), in drinking water by WHO and EPA [60,61]. The LOD comparison
+
2+
μ
μ
3
+
2+
M) and Cu (20.5
μ
μ
of RFC with other reported probes (Table 1) shows its great potency in
detecting only Al3 and Cu with the ability to discern the two metal
+
2+
ions by means of fluorescent enhancement.
3
.5. Association constant calculations
The association constants, K
determined using the Benesi-Hildebrand equations [67,68] as follow
of RFC towards Al3 and Cu were
+ 2+
a
Fig. 4. Fluorescence intensity of RFC (50
50
M) at λem = 588 nm.
μ
M) upon addition of various cations
1
1
1
(
μ
[
[
] + A
] + F
=
=
(1)
(2)
2
+
A ꢀ A
0
K (A ꢀ A ) Cu
max ꢀ A
0
a
max
0
Cu2 , titration experiment is conducted. Al and Cu titration against
RFC in MeCN/H O (9:1, v/v pH 7.5) was monitored using Fluorescence
and UV–vis spectra respectively. As shown in Fig. 5a), with increasing
+
3+
2+
1
1
1
2
3+
F ꢀ F
0
K (F ꢀ F ) Al
max ꢀ F
0
a
max
0
3
+
concentration of Al , the fluorescence intensity at 588 nm increased
A and A
0
is the absorbance of RFC solution in the presence and absence
accordingly. On the other hand, gradual addition of Cu2 causes an in-
+
2
+
of Cu respectively; Amax is the saturated absorbance in the presence of
excess amount of Cu2 ; [Cu ] is the concentration of metal ions added.
crease in the absorbance peak at 560 nm respectively.
+
2+
From the titration experiments conducted, the limit of detection
While F stands for the fluorescence intensity of RFC towards presence
Fig. 5. a) Fluorescence spectrum of Al at λ = 588 and b) UV–vis spectrum of Cu2+ at λ = 560 nm. Insets: Plot of changes of emission and absorbance intensity with
3
+
incremental addition of metal ions.
4

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
Table 1
3
+
2+
Comparison among reported chemosensors with RFC in respect to LOD of Al and Cu .
Sensor
Detection Method
Chemosensor based
Solvent
system
Metal (s) detected
LOD (
μ
M)
Ref.
ꢀ
2+
3+
2+
0.36 (Cu )
RD
F3
L
Fluorescence and Colorimetric
Fluorescence
Rhodamine
Fluorene
DMSO/H
MeCN
2
O
CN and Cu
[62]
[63]
[64]
3
+
3+
Cr and Al
0.31 (Al )
3
+
3+
2+
3+
2+
Fluorescence (Al and Cr only) and
Colorimetric
Rhodamine and
azobenzene
Thiazole
EtOH/H
2
O
Cu , Al and
1.00 (Cu ) 2.00
3
+
3+
Cr
(Al
)
3
+
L1&L2
Fluorescence
MeOH
Al
1.00 and 0.75
[65]
[66]
3
+
2+
3+
2+
1
Fluorescence (Fe only) and Colorimetric
Rhodamine
MeOH/H
MeCN/H
2
O
Cu , Al and
0.29 (Cu ) 0.01
3
+
3+
Fe
(Al
)
3
+
2+
3+
2+
RFC (proposed
sensor)
Fluorescence (Al only) and Colorimetric
Rhodamine and fluorene
2
O
Cu and Al
1.14 (Cu ) 0.12
–
3
+
(Al
)
and absence of Al3
+
.
ppm (4 eq. of Al ). Besides that, decrease in intensities and broadening
of peaks were also observed at 6.25 ppm, 6.27 ppm, 6.49 ppm, 6.55 ppm
3+
From the equation, by plotting a graph of 1/(F-F
0
) or 1/(A-A
0
) versus
+
3+
1
/[M ] (Fig. S4 (a) and (b)), a linear regression equation was obtained.
and 6.57 ppm (2 eq. of Al ) and 6.41 ppm, 6.62 ppm, 6.64 ppm, and
3
+
3+
Through the slope and intercept, the value of K
a
of RFC for Al and
6.70 ppm (4 eq. of Al ), corresponding to the xanthene protons. The
2
+
4
ꢀ 1
3
ꢀ 1
Cu were calculated as 1.83 × 10
M
and 4.56 × 10
M respec-
results indicate that the shifts and broadening of peaks are due to the
3
+
coordination of RFC with Al . The complete 1H NMR spectrum for
Fig. 8 could be found in the supplementary material (Fig. S5, S6 and S7).
Due to the paramagnetism of copper, the nuclear magnetic titration
was replaced with infrared spectrum (Fig. 9). From the partial IR spectra
3+
tively, suggesting that RFC has a higher binding capacity towards Al
than Cu2
+
.
3
.6. Job’s plot
Next, in determining the stoichiometry of RFC with Al3 and Cu
–
–
of RFC sensor, the stretching vibration absorption peaks of C O and
+
2+
–
–
ꢀ 1
ꢀ 1
,
CN
appeared at 1675 cm and 1614 cm respectively. Upon binding
3
+
2+
Job’s plot analysis was conducted. The molar concentration of Al and
Cu ranged from 0 to 1 with a fixed total concentration of RFC + M
100 M) in MeCN/H O (9:1, v/v, pH 7.5) at 560 nm. The results
revealed that maximum absorbance was achieved at molar fraction of
with Cu , the stretching vibration absorption peaks shifted to 1585
2
+
n+
ꢀ 1
cm . This shows that both the ketone and amide carbonyl group of RFC
are involved in the coordination with Cu2
+
(
μ
2
.
3
+
2+
0
.5 for both Al (Fig. 6a) and Cu (Fig. 6b), indicating that the ratio of
3.9. Reversibility
3
+
2+
RFC- Al and RFC- Cu complexes as 1:1 (Scheme 2).
Reversibility is an essential aspect for a chemical sensor for use in
practical application. The reversibility of RFC towards target analytes
3
.7. Competitive selectivity of RFC
3
+
2+
(
Al and Cu ) was tested by using EDTA. As depicted in Fig. S8a, upon
3
+
3+
To further investigate the selectivity of RFC in detecting Al and
+
addition of excessive amount of EDTA into the RFC + Al system,
decolourization of the solution occurs immediately (Fig. 10) followed by
the disappearance of absorbance peak at 560 nm. Subsequent addition
Cu2 , competitive selectivity of RFC (50
μ
M) in the presence of other
+
2+
2+
2+
3+
2+
+
2+
2+
2+
cations (Ag , Ca , Cd , Co , La , Mn , Na , Ni , Pb , Sn and
2
+
3+
Zn ) (50
μ
M) was studied using fluorescence and UV–vis spectroscopy
of Al into the system shows the recovery of the absorbance peak and
3
+
techniques. From Fig. 7, the recognition of Al by RFC is not signifi-
cantly influenced by other coexisting metal ions. However, in the case of
the solution changes back to purple colour. The same method was
applied for Cu2 (Fig. S8b) with repeatability of up to 10 times and up to
+
2
+
3+
Cu , aside from the significantly intensified absorbance in the presence
12 times for Al (Fig. S8a).
3
+
2+
of Al (Inset of Fig. 7) the detection of Cu by RFC is not affected by
other coexisting metal ions.
3.10. On site assay study
3
.8. Possible binding mechanism
To further elucidate the binding mode of RFC with Al3 , the H NMR
In order to test the potential of RFC for practical application, a paper
based on-site visual detection of Al3 and Cu by using the common
filter paper have been prepared. The test strips were prepared by soaking
the filter paper into RFC (25 mM) solution for 5 min and left to dry in air.
+
2+
+
1
titration experiment was carried out. As shown in Fig. 8, during gradual
addition of Al3 , the proton of C = N at 8.58 ppm becomes less intense,
+
It was then used to detect different concentration of Al and Cu in
aqueous media. As seen in Fig. 11a and b, it shows a gradual increase in
3+
2+
3
+
broadened and shifted downfield, to 8.59 ppm (2 eq. of Al ) and 8.73
Fig. 6. Job’s plot of RFC and Mn+ ([Mn+] + [RFC] =100
μ
M) at λ = 560 nm for a) Al3+ and b) Cu at 560 nm.
2+
5

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
3
+
2+
Scheme 2. Proposed binding mechanism of RFC with Al and Cu .
3
+
2
+
Fig. 7. The competitive selectivity of RFC (50 M) based on changes in fluorescence intensity for a) Al at 588 nm and absorbance changes for b) Cu at 560 nm in
μ
2
+
3+
the presence of various cations (50
μ
M). Inset: Absorbance changes for Cu in the presence of Al .
1
3+
Fig. 8. HNMR spectral changes of RFC upon addition of Al in CDCl
3
-d.
6

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
Fig. 11. Colorimetric changes of test strips upon addition of increasing metal
3
+
2+
ions for a) Al and b) Cu c) Changes in test-strips under the UV-lamp.
3
.11. In vitro cell growth inhibitory effects of the RFC
To evaluate the cytotoxicity of RFC sensor, cell viability was deter-
mined by MTT assay conducted on the human colorectal adenocarci-
noma (HT-29) and normal (CCD-18Co) cell lines with cisplatin as the
positive control (Table S2). As depicted in Fig. 12, at least 58 % of the
CCD-18Co cells and 47 % of HT-29 cells remained alive after incubation
for 72 h with RFC. On the other hand, at the highest concentration, the
cell viability was less than 20 % when both cells were treated with
3
+
cisplatin. Thus, the results suggest that RFC could be used to detect Al
and Cu2 in biological samples.
+
2
+
4. Conclusion
Fig. 9. Infrared spectra of RFC and RFC-Cu complex.
In summary, the current study reported a new rhodamine-based
chemosensor, RFC for simultaneous detection of Al3 and Cu . Not
only does RFC shows an “off-on’’ colorimetric changes via naked-eye
detection, it is also able to differentiate between the two metal ions
under the UV-lamp. Furthermore, RFC shows great sensitivity in
+
2+
Fig. 10. Colorimetric changes upon addition of EDTA and re-addition of
metal ions.
pink intensity of the test strips upon increasing concentration of ions
from 0 to 500
μ
M. By placing the test strips under the UV-lamp, the two
metal ions are easily distinguishable by appearance of bright orange
3
+
fluorescence for Al (Fig. 11c). These results show great applicability of
the test strips in detecting these metal ions in real water sample.
Fig. 12. Cytotoxic activity of RFC against HT-29 (a) and CCD-18Co (b) cell
lines. Cisplatin was used as positive control. Data is expressed as mean ± SD (n
=
3). *p < 0.05 indicates significant different from untreated control.
7

N.A.S. Pungut et al.
Journal of Photochemistry & Photobiology, A: Chemistry 414 (2021) 113290
detecting Al3 and Cu in an effective concentration as low as 0.23
+
2+
μM
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achieved and proven by adding excess EDTA into the solution containing
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gest great potentiality in detecting Al3 and Cu for environmental use
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Nur Amira Solehah Pungut: Writing- original draft, Validation,
Investigation, Visualization, Data curation, Methodology, Conceptuali-
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Kae Shin Sim: Writing - review & editing, Kong Wai Tan: Writing –
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Declaration of Competing Interest
[
21] T. Rasheed, C. Li, Y. Zhang, F. Nabeel, J. Peng, J. Qi, L. Gong, C. Yu, Rhodamine-
based multianalyte colorimetric probe with potentialities as on-site assay kit and in
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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This research was supported by Ministry of Higher Education for the
grant provided (FRGS/1/2019/STG01/UM/02/11) in completing the
project, and the collaboration of Ministry of Education and Universiti
Malaysia Sabah (UMS) for the scholarship provided. Special thanks to
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