A phenoxazine-based fluorescent chemosensor for dual channel detection of Cd2+ and CN? ions and its application to bio-imaging in live cells and zebrafish

Article abstract of DOI:10.1016/j.dyepig.2019.107828

A simple phenoxazine-based fluorescence chemosensor N-(4-((4-((5-oxo-5H-benzo [a]phenoxazin-6-yl)amino)phenyl)sulfonyl)phenyl)furan-2-carboxamide (NPC) was developed for discriminative detection of Cd²⁺ and CN^? ions. This easily available chemosensor was synthesized by adding a furan-2-carboxamide group to a phenoxazine fluorophore and a phenyl sulfonyl chelating site. The sensor NPC exhibited turn-on fluorescence emission for Cd²⁺, and the obtained NPC–Cd²⁺ complex detected CN^? via turn-off fluorescence response with 2:1 binding stoichiometry in CH₃CN/H₂O (99:1 v/v) medium by fluorescence spectroscopy. The detection limits of Cd²⁺ and CN^? were 0.60 μM and 0.145 μM, respectively, which were significantly lower than the World Health Organization guidelines. The proposed mechanism of sensor NPC was additionally supported by NMR, FT-IR analyses, DFT and a UV/visible titration studies. Furthermore, the sensor NPC has been successfully demonstrated as a biomarker for detecting Cd²⁺ in live cells and zebrafish larvae.

Full text of DOI:10.1016/j.dyepig.2019.107828

Journal Pre-proof
2+
A phenoxazine-based fluorescent chemosensor for dual channel detection of Cd
−
and CN ions and its application to bio-imaging in live cells and zebrafish
Palanisamy Ravichandiran, Anna Boguszewska-Czubara, Maciej Masłyk, Antony
Paulraj Bella, Princy Merlin Johnson, Sivakumar Allur Subramaniyan, Kwan Seob
Shim, Dong Jin Yoo
PII:
S0143-7208(19)31685-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.107828
DYPI 107828
Reference:
To appear in: Dyes and Pigments
Received Date: 18 July 2019
Revised Date: 12 August 2019
Accepted Date: 20 August 2019
Please cite this article as: Ravichandiran P, Boguszewska-Czubara A, Masłyk M, Bella AP, Johnson PM,
Subramaniyan SA, Shim KS, Yoo DJ, A phenoxazine-based fluorescent chemosensor for dual channel
2+
−
detection of Cd and CN ions and its application to bio-imaging in live cells and zebrafish, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107828.
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TOC GRAPHICS
The sensor NPC acts as a dual sensor for cadmium(II) and cycnide ions. The practical
applications of the sensor also successfully demonstrated to bio-imaging of live cells and
zebrafish.
1

A Phenoxazine-Based Fluorescent Chemosensor for Dual Channel Detection
of Cd2+ and CN- Ions and its Application to Bio-Imaging in Live Cells and
Zebrafish
Palanisamy Ravichandirana, Anna Boguszewska-Czubarab, Maciej Masłykc, Antony Paulraj
Bellad, Princy Merlin Johnsond, Sivakumar Allur Subramaniyane, Kwan Seob Shime, and Dong
Jin Yooa*
a
Department of Life Science, Department of Energy Storage/Conversion Engineering of
Graduate School, and Hydrogen and Fuel Cell Research Center, Chonbuk National University,
Jeollabuk-do 54896, Republic of Korea.
b
Department of Medical Chemistry, Medical University of Lublin, ul. Chodźki 4A, 20-093,
Lublin, Poland.
c Department of Molecular Biology, Faculty of Biotechnology and Environmental Sciences, The
John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland.
d PG and Research Department of chemistry, Bishop Heber College, Tiruchirappalli-620017,
Tamil Nadu, India.
e
Department of Animal Biotechnology, College of Agriculture and Life Sciences, Chonbuk
National University, Jeollabuk-do 54896, Republic of Korea.
*Correspondence
Prof. Dong Jin Yoo-PhD
Department of Life Science, Department of Energy Storage/Conversion Engineering of Graduate
School, Chonbuk National, University, Jeollabuk-do 54896, Republic of Korea
Tel: +82-(0)63-270-3608 /+82-(0)10-2110-2328 Fax: +82-(0)63-270-3909
E-mail: djyoo@jbnu.ac.kr; ravichandru55@gmail.com
1

ABSTRACT
simple
benzo[a]phenoxazin-6-yl)amino)phenyl)sulfonyl)phenyl)furan-2-carboxamide
A
phenoxazine-based
fluorescence
chemosensor
N-(4-((4-((5-oxo-5H-
(NPC) was
developed for discriminative detection of Cd2+ and CN- ions. This easily available chemosensor
was synthesized by adding a furan-2-carboxamide group to a phenoxazine fluorophore and a
phenyl sulfonyl chelating site. The sensor NPC exhibited turn-on fluorescence emission for Cd2+,
and the obtained NPC–Cd2+ complex detected CN- via turn-off fluorescence response with 2:1
binding stoichiometry in CH3CN/H2O (99:1 v/v) medium by fluorescence spectroscopy. The
detection limits of Cd2+ and CN- were 0.60 µM and 0.145 µM, respectively, which were
significantly lower than the World Health Organization guidelines. The proposed mechanism of
sensor NPC was additionally supported by NMR, FT-IR analyses, DFT and a UV/visible
titration studies. Furthermore, the sensor NPC has been successfully demonstrated as a
biomarker for detecting Cd2+ in live cells and zebrafish larvae.
KEYWORDS: Quinone, off-on-off sensor, fluorescence imaging, UV/visible absorption, DFT.
1. INTRODUCTION
In the last few decades, with the fast development of modern industries, heavy metal and anion
pollution has become a serious threat to human health and environmental safety [1,2] Among
various inorganic pollutants, cadmium (Cd) is a silver-white heavy metal with high toxicity that
is employed in various applications including phosphate fertilizers, Ni-Cd batteries, and
semiconducting quantum dots, resulting in severe environmental pollution [3,4]. According to
the agency for toxic substances and disease registry of the USA, the Environmental Protection
Agency, cadmium is number 7 among the top 275 harmful substances [5-7]. The World Health
Organization (WHO) recommended the optimum level of Cd in drinking water as 0.05 mg·m-3 to
2

avoid kidney impairment and other associated diseases [8]. The human body has no mechanism
to eliminate Cd; therefore, excess intake of the Cd present in water and air by drinking water,
eating food, and breathing cigarette smoke can cause a number of chronic disorders of the
gastrointestinal tract, liver, kidney, brain, and bone [9,10].
Additionally, cyanide ion (CN-) is particularly toxic to living systems due to its strong binding
affinity with cytochrome c oxidase, which prohibits the mitochondrial electron transport cycle
[11-13]. This inhibition causes vomiting, convulsions, loss of consciousness, and sudden death
[14]. However, cyanide is found in many foods and plants because of its tremendous usage in
gold mining, metallurgy, and electroplating industries [15]. According to the WHO, the
permissible level of cyanide ion concentrations in drinking water is less than 1.9 µM [16]. The
current research demonstrates that the cyanide level in the blood of human fire victims is
calculated at about 20 µM [17,18]. As a result, it is necessary to develop efficient and selective
sensors for recognition of Cd2+ and CN- ions.
Development and detection of environmentally and biologically important analytes have
become important goals of chemical sensors [19-22]. Numerous analytical techniques have been
developed in recent years for selective detection of particular analytes, such as high-performance
liquid chromatography, mass spectrometry, inductively coupled plasma atomic emission
spectrometry, atomic absorption spectroscopy, and electrochemical approaches [23-29].
However, these analytical methods suffer from disadvantages such as time-consuming processes,
relatively high cost, complicated operations, and sophisticated maintenance [30]. Accordingly,
development of simple and efficient sensing methods to monitor metals and anions is an essential
demand. In recent times, chemosensors and optical sensors have attracted wide interest owing to
their characteristic features of simplicity, high sensitivity, rapidity, low cost, easy operation, and
3

real-time detection [31-33]. Thanks to these merits, sensing of toxic analytes using organic
multifunctional probes has become an increasingly important new research focus [34,35].
Phenoxazines have been long utilized in organic electronics, but their usage in sensor chemistry
is very rare. Very few reports are available on phenoxazine chemosensors for selective detection
of toxic metals and anions. In addition, among various fluorogenic groups, phenoxazine exhibits
profound conformational changes. Thus, phenoxazine derivatives are perfect platforms for the
development of cation and anion recognition [36,37].
Fluorescence chemosensors are often employed to detect toxic ions because of their selectivity,
simplicity, and sensitivity. In 2012, Park et al. reported a naphthocoumarin-based fluorescent
sensor for dual detection of cyanide and cadmium ions [38]. Later in 2017, Shaily et al.
described a coumarin-chalcone hybrid as a colorimetric and fluorescence chemosensor for Cd2+
detection [7]. Recently in 2019, Wan et al. designated a quinolone-containing Schiff base
conjugate as a Cd2+ chemosensor [39]. In addition, Jia et al. reported 2-((6-
(diethylamino)quinolin-2-yl)methylene)malononitrile for selective detection of CN- ion [40].
Limited examples of chemosensors for dual channel detection of Cd2+ and CN- ions have been
reported [38,41,42] Nevertheless, these available fluorescence chemosensors suffer limitations,
including poor selectivity/sensitivity, weak fluorescence response, and lack of suitability for
biological systems.
In recent times, we reported the synthesis of phenoxazine-based chemosensor for selective
detection of Ba2+ ions [43]. We inspired by the efficiency of the phenoxazine probe, the
development of sensor probe for combined detection of Cd2+ and CN- ions in a polar aprotic
medium is reported in this article [44]. In this work, we present the synthesis and detection of a
fluorescence chemosensor for selective sensing of Cd2+ and CN- ions in CH3CN/H2O (99:1 v/v)
4

medium. To the best of our knowledge, the chemosensor N-(4-((4-((5-oxo-5H-
benzo[a]phenoxazin-6-yl)amino)phenyl)sulfonyl)phenyl)furan-2-carboxamide (NPC) is the first
model of a metal-generated TURN-ON and TURN-OFF type of fluorescence chemosensor for
Cd2+ and CN- ions. The coordination mechanism of the NPC–Cd2+ complex was confirmed by
NMR, UV/visible titrations, DFT and FT-IR analysis. Bio-imaging studies in live cells and
zebrafish were successfully performed to show the usefulness of NPC in biological systems.
2. EXPERIMENTAL SECTION
2.1. Synthesis of Sensor NPC
The synthetic approaches from our earlier reports with slight modifications were employed to
prepare the ligand NPC (Scheme 1) [45-47]. The synthetic methods and spectral characterization
of compounds 3 and 4 are available in our previous report [43]. The synthetic procedure and
spectral analysis of ligand NPC (1H NMR, 13C NMR and MS (ESI); Figs. S1 and S2 of the
Supporting Information, SI) are discussed in the SI.
2.2. Stock Solution Preparation of NPC and Metal Ions
All the fluorescence emission spectra were measured after adding metal ions and anions to
CH3CN/H2O (99:1 v/v) medium. An NPC stock solution (1.0 × 10-3 M) was prepared by
dissolving 0.0057 g of NPC in 10 mL of CH3CN/DMSO (1:1, v/v). Metal ions (1.0 × 10-1 M) of
K+, Na+, Ag+, Li+, Cs+, Ba2+, Cd2+, Mg2+, Cu2+, Zn2+, Ni2+, Ca2+, Sr2+, Co2+, Fe3+, and La3+ were
prepared in double distilled water and utilized for detection experiments. The anions (1.0 × 10-1
-
-
-
2-
-
M) CN-, ClO4 , NO3 , F-, H2PO4 , Cr2O7 , and BF4 were prepared in CH3CN and utilized for an
anion binding study. From the stock solutions, 50 µL of NPC (1.25 × 10-5 M) and 50 µL of metal
ions or anions (1.25 × 10-3 M) were diluted in 4 mL of CH3CN/H2O (99:1 v/v) solution that was
5

further used for metal ion or anion binding studies. Spectroscopic experiments were carried out
at room temperature (RT).
2.3. Synthesis of NPC-Cd2+ Complex for FT-IR Study
Ligand NPC (0.176 g, 0.01 M) and the metal salt cadmium acetate dihydrate (0.079 g, 0.01 M)
were dissolved in acetonitrile (CH3CN, 30 mL). At RT, the reaction mixture was stirred for 3 h.
The NPC-Cd2+ complex was precipitated in CH3CN and was then filtered by vacuum, carefully
washed with CH3CN (80 mL), and dried at 50 °C. FT-IR analysis was carried out for the isolated
NPC-Cd2+ complex by attenuated total reflection mode.
2.4. In Vitro Cytotoxicity Assessment by MTT Assay
MCF-7 cells (breast cancer) were used for MTT cytotoxicity study of NPC. MCF-7 cells were
cultivated in RPMI-1640 medium at 37 °C in a 5% CO2 atmosphere. After sufficient cells were
grown, they were collected and placed in a 96-well cell culture plate. Next, the cells were
incubated with NPC at 10 different doses from 0.195 µM to 100 µM for 24 h. After treatment of
cells with NPC, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,
5 mg/mL in phosphate buffer solution, PBS) was added. The MTT-treated cells were further
incubated for 4 h to form formazan crystals. The crystals were dissolved in 100 µL of DMSO,
and the optical density was measured at 590 nm. In this study, three independent experiments
were performed [48].
2.5. Bio-Imaging Studies in Live Cells
To evaluate the sensing application of NPC in living organisms, MCF-7 cells were used in this
study. The cells were cultivated in a 12-well culture plate at 5% CO2 atmosphere for 24 h. First, a
Cd2+ salt solution (6.25 µM) was introduced into the cells, and they were incubated for 30 min.
After the incubation period, the cells were thoroughly washed with PBS to remove excess Cd2+.
6

Then, the cells were incubated with NPC (12.5 µM) for 10 min. Following, the cells were
thoroughly washed with PBS solution to remove excess NPC. The treated MCF-7 cells were
fixed by 3.7% formaldehyde solution (pH 7.0), and fluorescence images were recorded by
fluorescence confocal microscopy in green and red channels installed at the Center for
University-Wide Research Facilities (CURF) at Chonbuk National University (CBNU, Jeonju,
South Korea) [43].
2.6. Bio-Imaging Studies in Zebrafish
Zebrafish larvae were maintained in E3 embryo media at 28 °C (5 mM/L NaCl, 0.33 mM/L
MgSO4, 0.33 mM/L CaCl2, and 0.17 mM/L KCl) on a 14 h light and 10 h dark cycle. The 5-day-
old baby zebrafish larvae were incubated with 12.5 µM of NPC for 20 min at 28 °C. After
treatment with NPC, the zebrafish were washed with PBS, and fluorescence images were
acquired. Subsequently, the same baby zebrafish were further incubated with 6.25 µM of Cd2+
ion for 15 min at 28 °C and washed with PBS solution to remove excess Cd2+ salt. Finally,
fluorescence imaging of treated zebrafish was carried out using confocal fluorescence
microscopy [49,50].
3. RESULTS AND DISCUSSION
3.1. Synthesis of Chemosensor NPC
Chemosensor NPC was prepared using simple and greener synthetic steps, as presented in
Scheme 1. In summary, the reaction of 2,3-dichloro-1,4-naphthoquinone 1 and 4-aminophenyl
sulfone 2 was accomplished in the green solvent water via a Michael-like addition that afforded
compound 3. Next, compound 3 was utilized for cyclization reaction with 2-aminophenol in
CH3CN and K2CO3 to give compound 4 with good yield. Finally, cyclized product 4 was
7

involved in a reaction with 2-furoyl chloride in acetone to give the preferred chemosensor NPC
with good yield.
Scheme 1. Synthesis of chemosensor NPC
3.2. Fluorescence Response of Chemosensor NPC for Detection of Cd2+
The synthesized compound 4 and chemosensor NPC were evaluated for their solvatochromic
properties, showing three absorption maxima at 230, 325, and 530 nm due to their π–π*
transition. In addition, a highly intensified absorption peak was found in CH3CN/H2O (99:1 v/v)
medium (Figs. S3 and S4). Therefore, the emission wavelength (530 nm) and the solvent
medium (CH3CN/H2O) were carefully chosen for further fluorescence emission studies. First,
compounds 3 and 4 were employed for a selectivity study with the metal ions K+, Na+, Ag+, Li+,
Cs+, Ba2+, Cd2+, Mg2+, Cu2+, Zn2+, Ni2+, Fe2+, Ca2+, Sr2+, Co2+, Fe3+, and La3+. Compound 3 did
not show any characteristic fluorescence emission peak at the fixed wavelength. In contrast,
compound 4 showed sensing properties for Cd2+ and Ba2+ ions along with interference from
8

other metal ions. The selectivity of metal ions with compound 4 was not appreciable; this may be
due to lack of appropriate amide moieties and furan ring systems in the phenoxazine unit (Fig.
S5). Thus, the chemosensor NPC has been synthesized and investigated for its sensing behavior.
No significant changes were found in monomer fluorescence emission intensity and peak
position of the sensor NPC with the metal ions (K+, Na+, Ag+, Li+, Cs+, Ba2+, Mg2+, Cu2+, Zn2+,
Ni2+, Fe2+, Ca2+, Sr2+, Co2+, Fe3+, and La3+). The obtained weak fluorescence emission due to
isomerization of C=N and weak intramolecular charge transfer (ICT) process occurred in sensor
NPC. Fascinatingly, 130-fold increased fluorescence intensity was acquired upon the addition of
Cd2+ (Fig. 1a). This fluorescence enrichment was due to charge transfer between the lone
electron pair in the secondary amine and amide groups (–NH) of the phenoxazine moiety, which
induces strong ICT. In addition, the polar solvent mixture CH3CN:H2O (99:1) primarily prefers
these mechanistic pathways. When ligand NPC was synthesized by adding a furan-2-
carboxamide group to phenoxazine fluorophore and a phenyl sulfonyl chelating site, it was more
selective for Cd2+ rather than Ba2+ and other co-existing metal ions. To confirm the binding
selectivity of chemosensor NPC to detect Cd2+, the complexometric titration experiment was
carried out with coexistence of other metal ions. As presented in Fig. 1b, no significant changes
were perceived in fluorescence intensity. Thus, these results indicated the excellent binding
selectivity of NPC toward Cd2+ and poor binding ability for other metal ions.
9

Cd2+
(a)
800
600
400
200
0
NPC, NPC + K+, Na+,
Ag+, Li+, Cs+, Mg2+,
Cu2+, Zn2+, Ni2+, Fe2+,
Ca2+, Sr2+, Co2+, Fe3+,
La3+
Fe2+
Ba2+
510
520
530
540
550
Wavelength (nm)
1000
800
600
400
200
0
NPC + Metal ions
NPC + Cd (II) + Other metal ions
(b)
NPC Cd K (I) Na Ag Li (I) Cs Ba Mg Cu Zn Ni Fe Ca Sr Co La
(II) (I) (I) (I) (II) (II) (II) (II) (II) (II) (II) (II) (II) (III)
10

Fig. 1. (a) Fluorescence emission spectra of chemosensor NPC (1.25ꢀ×ꢀ10-5ꢀM) in the presence of
various metal ions (1.25ꢀ×ꢀ10-3ꢀM) in CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm; (b)
Fluorescence emission of NPC (1.25ꢀ×ꢀ10-5ꢀM) in the presence of Cd2+ (1.25ꢀ×ꢀ10-3ꢀM) and
added different metal ions (1.25ꢀ×ꢀ10-3ꢀM) in CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm.
(For the understanding of the references to color in this figure legend, the reader is referred to the
web version of this article)
To gain deep insight into the sensing behavior of NPC to Cd2+ at the molecular level,
fluorescence titration experiments in CH3CN/H2O (99:1 v/v) medium were performed. As shown
in Fig. 2, upon increasing addition of Cd2+ (0–1.25ꢀ×ꢀ10-3ꢀM) to the NPC solution, a gradual
increment in fluorescence intensity was noticed. The increased fluorescence emission was
arrested after adding 100 equivalents of Cd2+ due to the NPC–Cd2+ complex reaching saturation
in solution medium. If Cd2+ concentration was limited to 1.25ꢀ×ꢀ10-3ꢀM or lower, NPC presented
a linear curve (R2 = 0.9947, Fig. S6).
11

100 equiv.
500
400
300
200
100
0
Cd2+
0 equiv.
510
520
530
540
550
Wavelength (nm)
Fig. 2. Fluorescence titration of NPC (1.25ꢀ×ꢀ10-5ꢀM) with the increasing amount of Cd2+ (0–
1.25ꢀ×ꢀ10-3ꢀM) in CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm.
To calculate the detection limit, the 3σ/k formula was used, where σ is the standard deviation
of the blank measurement, and k is the slope of the intensity against the concentration of the
sample [27]. The calculated detection limit was 0.60 µM, which is lower than the WHO’s
recommended limit for Cd2+ in drinking water (0.05 mg·m-3). The obtained Job's plot analysis
confirmed a 2:1 stoichiometric complex in NPC–Cd2+ (Fig. S7). The binding constant (Ka) of
NPC–Cd2+ complex was calculated by using fluorescence titration spectrums (from Fig. 2) using
a revised Benesi-Hildebrand equation:[51] Io/I-Io = (a/b-a) (1/Ka[C]+1), where Io and I are the
fluorescence intensities of NPC at 530 nm in the absence and existence of Cd2+, respectively; a
and b are the constants; Ka is the association constant; and [C] is the concentration of Cd2+ [52].
12

The calculated binding constant was 1.36ꢀ×ꢀ106ꢀM-2 from the plot of 1/(I-Io) against 1/[C] of Cd2+;
this indicates good binding affinity in the NPC–Cd2+ complex (Fig. 3).
0.2
0.15
0.1
y = 3E-09x - 0.0041
R² = 0.9945
0.05
0
0
20000000
40000000
1/[Cd2+]2, M-2
60000000
Fig. 3. Benesi-Hildebrand plot for 2:1 complex of NPC (1.25ꢀ×ꢀ10-5ꢀM) with Cd2+ (0–1.25ꢀ×ꢀ10-
3ꢀM) in CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm.
For practical application of the chemosensor NPC in biological systems, reversibility is
essential. Hence, a reversibility study was carried out in CH3CN/H2O (99:1 v/v) medium at λem
=
530 nm between NPC (1.25ꢀ×ꢀ10-5ꢀM) and Cd2+ (1.25ꢀ×ꢀ10-3ꢀM) upon addition of EDTA
(30 equiv.) solution. The added EDTA was chelated by the NPC–Cd2+ complex and quenched
the fluorescence intensity, similar to the original intensity of free ligand NPC. This result
indicates that the sensing process is reversible (Fig. 4). Therefore, the present chemosensor NPC
can be utilized as a probe to detect Cd2+ in real sample analysis and in biological systems.
13

1000
800
600
400
200
0
NPC
NPC + Cd (II)
NPC + Cd (II) + EDTA
505
515
525
535
545
555
Wavelength (nm)
Fig. 4. Reversibility study of NPC (1.25ꢀ×ꢀ10-5ꢀM) and Cd2+ (1.25ꢀ×ꢀ10-3ꢀM) complex upon
addition of EDTA solution (30 equiv.) in CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm.
3.3. NMR Titration Study
1
To obtain detailed information on the sensing mechanism of the NPC–Cd2+ complex, H NMR
titration experiments were carried out in DMSO-d6. As presented in Fig. 5a, the proton signals of
the secondary amine –NH and amide –NH groups in ligand NPC appeared at δꢀ=ꢀ8.6593 ppm
and 10.5611 ppm, respectively. With addition of increasing amounts of Cd2+ to NPC solution, the
signals of secondary amine –NH and amide –NH groups showed significant downfield shifts
from δꢀ=ꢀ8.6593 to δꢀ=ꢀ8.6657ꢀppm and 10.5611 to 10.5921 ppm, respectively, in the presence of
5.0 equiv. of Cd2+. The signals of aromatic protons also underwent small downfield shifts; no
further chemical shifts were obtained after addition of 5.0 equiv. of Cd2+. In addition, 13C NMR
titration experiments were performed to confirm the participation of amide –C=O and
14

phenoxazine –C=O moieties of ligand NPC. The carbonyl signals were obtained at 156.3793 and
179.2251 ppm for the respective functional groups. When adding increased amounts of Cd2+ to
DMSO-d6 containing NPC, notable downfield shifts were observed from 156.3793 to 156.4353
ppm and 179.2251 to 179.2674 ppm for the amide –C=O and phenoxazine ketone moieties,
respectively (Fig. 5b). In addition, no additional chemical shifts were observed after addition of 5
equiv. of Cd2+ due to saturation of the NPC–Cd2+ complex in DMSO-d6. These combined NMR
titration results indicate that the secondary amine –NH, amide –NH, amide –C=O, and
phenoxazine –C=O moieties of NPC were involved in complex formation with Cd2+. In addition,
these results revealed that the stoichiometric ratio of the NPC–Cd2+ complex was 2:1.
15

16

Fig. 5. (a) 1H NMR titration spectrum of NPC with (a) 0.0 equiv., (b) 1.0 equiv., (c) 2.0 equiv.,
(d) 3.0 equiv., (e) 4.0 equiv., (f) 5.0 equiv., of Cd2+; (b) 13C NMR titration spectrum of NPC with
17

the addition of (a) 0.0 equiv., (b) 1.0 equiv., (c) 2.0 equiv., (d) 3.0 equiv., (e) 4.0 equiv., (f) 5.0
equiv., of Cd2+ (solvent: DMSO-d6).
3.4. FT-IR Spectral Study
To investigate the in-depth mechanism of the NPC–Cd2+ complex, FT-IR analysis was
performed. As shown in Fig. 6, the IR spectrum of sensor NPC showed specific signals at 1493
cm-1 (amide –NH), 1682 cm-1 (amide –C=O), 1691 cm-1 (phenoxazine –C=O), 3275 cm-1
(secondary amine –NH), and 3361 cm-1 (amide –NH). The signal at 1443 cm-1 was attributed to
OAc- absorption from Cd(OAc)2·2H2O. Upon addition of two equivalents of Cd2+ into the
chemosensor NPC, significant blue shifts were obtained in the signals of amide –C=O (1667 cm-
1), phenoxazine –C=O (1681 cm-1), secondary amine –NH (3091 cm-1), and amide –NH (3332
cm-1). In contrast, a red shift was noted for the amide –NH (1501 cm-1) group. In addition, a
more intense and visible peak was obtained for the amide –NH group. These findings indicate
possible metal coordination through the amide –C=O, phenoxazine –C=O, secondary amine –
NH, and amide –NH moieties. The mechanism of the NPC–Cd2+ complex can be described by
Pearson’s hard-soft acid-base theory, wherein nitrogen cores fascinate towards heavy metal Cd2+
[53]. Furthermore, due to the oxophilic nature of Cd2+, –C=O groups attract Cd2+ ions [54].
18

Fig. 6. FT-IR spectrums of Cd(CH3COO)2.2H2O, NPC and NPC–Cd2+ complex.
3.5. Electrochemical Study of NPC
To evaluate the binding mechanism of the NPC–Cd2+ complex by electrochemical methods, a
cyclic voltammetry (CV) study was carried out, in which CH3CN was used as a solvent and
TBABF4 (tetrabutylammonium tetrafluoroborate, 0.1 M) was employed as a electrolyte medium
19

(Fig. S8). Anodic (Epa) and cathodic (Epc) peaks were obtained at a scan rate of 100 mV·s-1 [55].
From the obtained CV results, one anodic and one cathodic peaks were found for NPC (1.25 ×
10-5 M) at Epa = -1.147 V and Epc = -1.238 V, separately. While adding Cd2+ (1.25 × 10-5 M) to
the NPC solution, the current densities of both peaks were significantly inhibited, and the peak
potentials were moved to Epa = -1.077 V and Epc = -1.191 V, respectively. Hence, this
electrochemical study clearly revealed an effective binding affinity of in the NPC–Cd2+ complex.
3.6. Fluorescence Responses of NPC–Cd2+ Complex toward CN-
Considering the strong binding nature of the NPC–Cd2+ complex, it was anticipated to act as a
-
-
-
2-
chemosensor and an anion sensor. Hence, anions of CN-, ClO4 , NO3 , F-, H2PO4 , Cr2O7 , and
BF4 (in the form of tetrabutylammonium salts, 1.25ꢀ×ꢀ10-3ꢀM) were tested with the NPC–Cd2+
-
complex; only CN- was noticeably detected in a manner that led to a major decrease in
fluorescence intensity (Fig. 7a). Seemingly, the NPC–Cd2+ complex showed a fluorescent “turn-
off” CN- sensor distinct from its behavior when tested with the other examined anions. These
results indicate that the NPC–Cd2+ complex is more selective for detection of cyanide among
various anions.
To confirm the selectivity and practical applications of the NPC–Cd2+ complex as a selective
chemosensor for CN-, an anion competitive experiment was conducted. The study was conducted
with the NPC–Cd2+ complex in the presence of CN- and other anions (1.25ꢀ×ꢀ10-3ꢀM). The
obtained results show no significant changes in quenching of the fluorescence spectrum of
chemosensor NPC–Cd2+ with CN- by successive addition of other rival anions. Hence, these
results indicate that the NPC–Cd2+ complex acts as an excellent chemosensor for CN- detection
(Fig. 7b).
20

1000
800
600
400
200
0
NPC + Cd (II) + other anions
NPC + Cd (II) + cyanide + other anions
(b)
-
-
-
CN-
F-
Cr2O7
-
-
Cd (II)
NO3
H2PO4
BF4
ClO4
21

Fig. 7. (a) Fluorescence spectrum of chemosensor NPC (1.25ꢀ×ꢀ10-5ꢀM) in the presence of Cd2+
(1.25ꢀ×ꢀ10-3ꢀM) with the addition of various anions (1.25ꢀ×ꢀ10-3ꢀM) in CH3CN/H2O (99:1 v/v)
medium; λem = 530ꢀnm; (b) Anion competitive study of chemosensor NPC (1.25ꢀ×ꢀ10-5ꢀM) in the
presence of Cd2+ (1.25ꢀ×ꢀ10-3ꢀM), CN- (1.25ꢀ×ꢀ10-3ꢀM) and other anions (1.25ꢀ×ꢀ10-3ꢀM) in
CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm. (For the understanding of the references to color
in this figure legend, the reader is referred to the web version of this article)
Moreover, to understand the Cd2+ displacement property, fluorescence titration studies of
NPC–Cd2+ in the presence and absence of various concentrations of CN- were conducted. As
presented in Fig. 8a, upon addition of increasing concentrations of CN- to the NPC–Cd2+
complex, the fluorescence emission intensity of NPC–Cd2+ system was gradually quenched. The
fluorescence intensity was quenched by 96% and became identical to the fluorescence
wavelength of free NPC with 50 equiv. of CN-. No further fluorescence quenching emission was
obtained with the addition of over 50 equiv. of CN-, indicating that the NPC–Cd2+ solution
reached saturation. The Job’s plot analysis showed a maximum intensity at about 0.4 mol
fractions, demonstrating that NPC–Cd2+ formed a 2:1 complex with CN- (Fig. S9). In addition,
the detection limit of NPC–Cd2+ with CN-, which was calculated by the above method, was
0.145 µM, which is 138-fold lower than the WHO’s recommendation of the acceptable level of
CN- in drinking water (R2 = 0.9928, Fig. S10). Furthermore, the binding constant was calculated
using the above modified Benesi-Hildebrand equation. The calculated binding constant was Ka =
1.30ꢀ×ꢀ107ꢀM-2, and this binding constant indicates good binding attraction between NPC–Cd2+
and CN- systems (Fig. S11).
Reversibility is an essential need of any chemical sensor; thus, the reversibility of chemosensor
NPC was evaluated by five titration cycles of continuous additions of Cd2+ and CN- to NPC
22

solution. As shown in Fig. 8b, the reversibility of the sensor NPC showed alternative increasing
and decreasing of fluorescence emission intensity. This result indicates that the receptor NPC–
Cd2+ could be employed as a good fluorescence chemosensor for sensing of CN- in the fields of
environmental and biological systems.
1000
NPC + Cd (II) + other anions
NPC + Cd (II) + cyanide + other anions
(b)
800
600
400
200
0
-
-
-
-
CN-
F-
Cr2O7
-
Cd (II)
NO3
H2PO4
BF4
ClO4
23

1000
800
600
400
200
0
(b)
Cd2+
NPC
CN-
0
1
2
3
4
5
6
7
8
9
10 11
Number of addition
Fig. 8. (a) Fluorescence spectrum of sensor NPC (1.25ꢀ×ꢀ10-5ꢀM) in the presence of Cd2+
(1.25ꢀ×ꢀ10-3ꢀM) with the addition of different concentrations of CN- (0–6.25ꢀ×ꢀ10-4ꢀM) in
CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm; (b) Change in fluorescence emission of NPC
(1.25ꢀ×ꢀ10-5ꢀM) on the alternative addition of Cd2+ and CN- (each of 1.25ꢀ×ꢀ10-3ꢀM) in
CH3CN/H2O (99:1 v/v) medium; λem = 530ꢀnm.
3.7. Proposed Binding Mechanism
Based on the results obtained, a conceivable binding mechanism was suggested, as shown in
Scheme 2. An intramolecular charge transfer (ICT) process occurred in the phenoxazine
fluorophore, and the additional energy was shared to the phenyl sulfone unit, which induced the
strong ICT process shown by the weak fluorescence emission intensity. However, upon
complexation of NPC with Cd2+, maximum vibrational energy occurred due to the lone pair of
24

electrons available from nitrogen and oxygen atoms in the sensor NPC that result in the
inhibition of ICT process and enhances the fluorescence emission intensity. Fluorescence
enhancement and quenching were obtained with added Cd2+ and CN- ions due to respective
saturation, which affords mechanical rigidity of NPC–Cd2+ complex and free ligand NPC.
Therefore, selective recognition of cadmium and cyanide ions became possible with the sensor
NPC [56]. This mechanism was further theoretically and experimentally supported by DFT and
UV/visible absorption titration studies. The molecular orbital geometry of the ligand NPC was
optimized by B3LYP/6-31G (d,p) level, using Gaussian 09 W program. As shown in Fig. 9, the
energy was distributed for the ligand NPC at phenoxazine fluorophore and phenyl sulfone units
at LUMO energy level, however, this energy distribution was restricted at sulfone unit in HOMO
energy level (∆E = 2.50 eV). This confirms that the ICT process occurred in free NPC. The ICT
inhibition process was confirmed by UV/visible absorption titration of NPC with an added
amount of Cd2+. The absorption peak intensity was gradually increased with the increasing
addition of Cd2+ in CH3CN/H2O (99:1, v/v). Besides, no characteristic blueshift or redshift were
noticed in the peak position (R2 = 0.992, Figs. S12 and S13). Thus, these obtained cumulative
results indicate that the NPC–Cd2+ complex operate via ICT and inhibited ICT mechanistic
pathways.
25

Fig. 9. The optimized geometry of NPC (A); LUMO (B) and HOMO (C) energy gaps of NPC.
26

ICT “off”
ICT “on”
Fluorescence “turn-off”
ICT “off”
Fluorescence “turn-on”
Scheme 2. Proposed binding mechanism of chemosensor NPC with Cd2+ and CN- ions.
3.8. Bio-Imaging Studies in Live Cells and Zebrafish
Furthermore, to explore the practical applications of ligand NPC in a biological system, its
toxicity to cancer cells was evaluated by MTT assay method. MCF-7 cells were chosen for this
study, and the results are shown in Fig. S14. Ten various concentrations of NPC (0.195 µM to
100 µM) were administered to MCF-7 cells, and the survival rates of the cells were above 95%
for concentrations of 0.195 to 12.5 µM. However, some of the higher concentrations of NPC (25
µM to 100 µM) were moderately toxic to the cancer cells. Thus, the obtained MTT assay results
suggest that the ligand NPC has low toxicity to living cells.
Based on the MTT toxicity profile of NPC, three concentrations (3.125 µM, 6.25 µM, and 12.5
µM) were selected for bio-imaging study in MCF-7 cells. As shown in Fig. 10a, a weak or no
fluorescence signal was detected when the cancer cells were separately incubated with metal
27

solution of Cd2+ and sensor probe NPC. Fascinatingly, when MCF-7 cells were incubated with
Cd2+ (6.25 µM) and then NPC (12.5 µM), the MCF-7 cell membranes treated with NPC–Cd2+
complex exhibited a dual channel emission of strong green and red fluorescence. Among the
three NPC concentrations studied for bio-imaging properties, 12.5 µM of NPC showed better
fluorescence imaging than the other concentrations (3.125 µM and 6.25 µM, Fig. S15).
Therefore, the bio-imaging studies indicated that the chemosensor NPC could be an excellent
sensor for fluorescence imaging of Cd2+ ion in MCF-7 cells, and that the sensor probe NPC has
possible application in metal ion analysis of biological samples (Table S1).
In recent times, zebrafish has been documented as a most effective animal model for bio-
imaging studies due to its transparency during all stages of embryonic development [57]. Hence,
our in vivo bio-imaging study of sensor probe NPC was performed using 5-day-old zebrafish
larvae. As shown in Fig. 10b, when zebrafish larvae were separately incubated with NPC (12.5
µM) or Cd2+ (6.25 µM), no significant fluorescence emission was observed. In addition,
zebrafish larvae further incubated with both NPC and Cd2+ salt solution showed strong
fluorescence emission. These results indicate that chemosensor NPC can be employed for
sensitive and selective detection of Cd2+ in animal models.
28