A coumarin-derived Cu2?+-fluorescent chemosensor and its direct application in aqueous media

Article abstract of DOI:10.1016/j.saa.2017.07.047

A novel coumarin-based receptor bearing a benzohydrazide (FCBH) was developed as a fluorescent chemosensor with high selectivity toward Cu^2?+. The sensor was successfully applied to the monitoring of Cu^2?+ in aqueous solution. After the addition of Cu^2?+ to FCBH, the color of the solution changed from greenish-yellow to red, and the absorption band at 457?nm red-shifted to 517?nm. The fluorescent green color of FCBH disappeared and the fluorescence emission was completely quenched in the presence of Cu^2?+. Upon the addition of Cu^2?+, deprotonation of FCBH occurred, and a 1:1 metal-ligand complex formed. DFT theoretical investigation was carried out to understand the behavior of the sensing probe toward Cu^2?+. Additionally, the quenched fluorescence of the FCBH-Cu^2?+ complex was restored upon the addition of CN^? ions. The possible sensing mechanism of FCBH toward Cu^2?+ was derived from experimental and theoretical examinations.

Full text of DOI:10.1016/j.saa.2017.07.047

Accepted Manuscript
A coumarin-derived Cu2+-fluorescent chemosensor and its direct
application in aqueous media
Naveen Mergu, Myeongjin Kim, Young-A. Son
PII:
S1386-1425(17)30607-8
doi: 10.1016/j.saa.2017.07.047
SAA 15334
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Accepted date:
22 May 2017
12 July 2017
24 July 2017
Please cite this article as: Naveen Mergu, Myeongjin Kim, Young-A. Son , A coumarin-
derived Cu2+-fluorescent chemosensor and its direct application in aqueous media,
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ACCEPTED MANUSCRIPT
2
+
A coumarin-derived Cu -fluorescent chemosensor and its direct application
in aqueous media
1
1
Naveen Mergu , Myeongjin Kim , Young-A. Son*
Department of Advanced Organic Materials Engineering, Chungnam National University, 220
Gung-dong, Yuseong-gu, Daejeon 305-764, South Korea
*
Corresponding author. Tel.: +82 42 821 6620; Fax: +82 42 821 8870.
E-mail addresses: yason@cnu.ac.kr (Y.-A. Son).
1
These authors contributed equally to this work.

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Abstract
A novel coumarin-based receptor bearing a benzohydrazide (FCBH) was developed as a
2
+
fluorescent chemosensor with high selectivity toward Cu . The sensor was successfully applied
2+
2+
to the monitoring of Cu in aqueous solution. After the addition of Cu to FCBH, the color of
the solution changed from greenish-yellow to red, and the absorption band at 457 nm red-shifted
to 517 nm. The fluorescent green color of FCBH disappeared and the fluorescence emission was
2
+
2+
completely quenched in the presence of Cu . Upon the addition of Cu , deprotonation of
FCBH occurred, and a 1:1 metal-ligand complex formed. DFT theoretical investigation was
2
+
carried out to understand the behavior of the sensing probe toward Cu . Additionally, the
2
+
−
quenched fluorescence of the FCBH-Cu complex was restored upon the addition of CN ions.
2
+
The possible sensing mechanism of FCBH toward Cu was derived from experimental and
theoretical examinations.
Keywords: Coumarin, Fluorescence, Chemosensor, Copper, Cyanide, Aqueous media
1
. Introduction
In the past few years, the development of fluorescent chemosensors for ion and molecular
recognition has received increased attention due to their potential applications in biology,
medicinal, clinical, catalysis and environmental monitoring [1-5]. Of the many types of
techniques, fluorescence sensing has several advantages over other methods such as atomic
absorption spectroscopy [6], inductively coupled plasma spectroscopy [7,8], neutron activation
analysis [9], chromatography [10], voltammetry [11] and others [12-16] as it possesses intrinsic
sensitivity and selectivity, low-cost, convenience, and real-time monitoring with fast response

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2
+
times [17-20]. The recognition of transition and heavy metal ions, notably of Cu , is particularly
attractive to current researchers, as it is an essential nutrient for life, and both its deficiency and
excesses are associated with various disorders [21–23].
Copper is the third most abundant metal among the essential heavy metal ions in the
human body [24] and it is crucial in a variety of fundamental physiological processes in
organisms [25]. Additionally, it is an essential trace element in biological systems, and its
importance may be attributed to its redox nature [26]. Extreme ingestion of copper is harmful
due to its ability to generate reactive oxygen species, and it can interfere in cellular metabolism
[
27]. In addition, unregulated copper can cause various neurodegenerative disorders, including
Wilson’s disease [28], Alzheimer’s disease [29], Parkinson’s disease [30] and Menkes disease
31]. According to the US Environmental Protection Agency, the limit of tolerable concentration
[
2
+
of Cu ions in drinking water is 20 µM. Therefore, the detection of copper has been raised as an
2
+
important consideration. However, while many fluorescent sensing systems for Cu -selective
detection have been reported [32-36], some of them have exhibited low sensitivity, poor
selectivity and low water solubility. Therefore, the development of a highly sensitive and
selective fluorescent probe for the detection of copper ions with a fast response time in aqueous
solution is necessary.
Coumarin derivatives are widely utilized as signaling units in chemosensors due to their
excellent chromogenic and fluorogenic properties [37-40]. In our recent study, a 3-
formylcoumarin benzoylhydrazone (FCBH)-derived fluorescent probe was synthesized and
characterized, and its metal ion-recognition properties were investigated for the first time. The
FCBH probe exhibited highly selective colorimetric and fluorescence quenching recognition of

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2
+
Cu . Electrospun nanofiber test strips were prepared for the detection of copper ions in aqueous-
alcoholic media.
2
. Experimental
2
.1. Reagents and apparatus
All the reagents for the synthesis of the FCBH chemosensor and metal salts (nitrates and
acetates) for the analysis were obtained commercially and used as received. FT-IR spectra were
1
13
recorded on an ALPHA-P spectrometer. H and C NMR spectra were performed on a Bruker at
3
00 and 600 MHz using TMS as an internal standard, CDCl and DMSO-d as the solvents. The
3
6
mass spectra were obtained on a Bruker-micrOTOF QII mass spectrometer. The UV-Vis
absorption spectra and the fluorescence emission spectra were recorded using an Agilent 8453
spectrophotometer and RF-5301PC spectrofluorophotometer, respectively. The excitation and
emission slit widths were 3.0 and 1.5 nm, respectively, for the emission studies. DFT
calculations were carried out using the Gaussian 09 program at the B3LYP level using the 6-
3
1G** basis set [41]. The geometry optimization was carried out using the M06 [42] hybrid meta
exchange correlation density functional at the 6-31G (d,p) level of theory. We performed a
population analysis to achieve electron density distribution isosurfaces. SEM images of
nanofibers were obtained on a TESCAN-Vega II/LSU. Fluorescence imaging was performed
with the Zeiss LSM 5 Live confocal microscope system.
2
.2. Synthesis and characterization
A coumarin-derived receptor (FCBH) was designed and synthesized following the route
outlined in Scheme 1.

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Scheme 1. Synthesis of (E)-N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-3,4,5-
trimethoxybenzohydrazide (FCBH).
Compound 1a was prepared according to the literature method [43]
7
-(Diethylamino)-2-oxo-2H-chromene-3-carbaldehyde (1b): Anhydrous DMF (2 mL) was
added dropwise to POCl (2 mL) at 0 °C and stirred for 30 min to form a white solid. Then, this
3
was combined with a solution of 1a (1.08 g, 5 mmol) in DMF (10 mL) to create a scarlet
suspension. The mixture was stirred at 70 °C for 16 h. After cooling to room temperature, the

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mixture was poured into 100 mL of ice water. Upon addition of NaOH (40%) solution to adjust
the pH to 5, a large amount of precipitate appeared. The product was filtered, thoroughly
washed with water, dried and the residue was purified by column chromatography using
DCM/Hexane (90:10) to give the desired compound 1b as an orange powder (0.75 g, 61%). 1b:
−
1
1
FT-IR (KBr), ν (cm ): 1129, 1256, 1349, 1503, 1569, 1712, 2970; H NMR (CDCl , 600 MHz),
3
δ (ppm): 1.25 (6H, t, J = 7.2 Hz), 3.47 (4H, q, J =7.2 Hz), 6.48 (1H, d, J =2.4 Hz), 6.63 (1H, dd,
1
3
J = 9.0, 2.4 Hz), 7.40 (1H, d, J = 9.0 Hz), 8.24 (1H, s), 10.12 (1H, s); C NMR (CDCl , 600
3
MHz), δ (ppm): 12.42, 45.25, 97.14, 108.21, 110.16, 114.31, 132.47, 145.33, 153.44, 158.91,
+
1
3
2
61.84, 187.90. ESI-MS m/z: Calcd for C H NO (M+Na) : 268.0950, found: 268.0971.
1
4
15
3
,4,5-Trimethoxybenzohydrazide (2a): A mixture of methyl 3,4,5-trimethoxybenzoate (0.45 g,
mmol) and hydrazine hydrate (55%, 0.64 g, 20 mmol) in 20 mL of ethanol was refluxed for 12
h. After completion of the reaction, the EtOH was evaporated under reduced pressure. Hexane
was added into the crude mixture, and the obtained precipitate was filtered and washed with
−
1
hexane to afford 2a as a white solid (0.4 g, 89%). 2a: FT-IR (KBr), ν (cm ): 1126, 1230, 1341,
1
1
581, 3334; H NMR (CDCl , 600 MHz), δ (ppm): 3.87 (3H, s), 3.88 (6H, s), 6.98 (2H, s), 7.54
3
13
(
1H, s); C NMR (CDCl , 600 MHz), δ (ppm): 56.3, 60.9, 104.3, 153.3. ESI-MS m/z: Calcd for
3
+
C H N O (M+Na) : 249.0851, found: 249.0859.
10
14
2
4
(
E)-N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-3,4,5-
trimethoxybenzohydrazide (FCBH): To a solution of 7-diethylamino-3-formylcoumarin (1b,
45 mg, 1 mmol) in ethanol (20 mL), 3,4,5-trimethoxybenzohydrazide (2a, 226 mg, 1 mmol)
2
was added. The resulting solution was refluxed for 6 h. Then, the solvent was removed and the
residue was purified by column chromatography using DCM/MeOH (95:5) to obtain compound
−
1
FCBH as an orange powder (360 mg, 79%). FCBH: FT-IR (KBr), ν (cm ): 1072, 1121, 1185,

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1
1
226, 1328, 1413, 1501, 1574, 1710, 2969; H NMR (CDCl , 600 MHz), δ (ppm): 1.22 (6H, t, J
3
=
7.2 Hz), 3.43 (4H, q, J =7.2 Hz), 3.88 (3H, s), 3.92 (6H, s), 6.43 (1H, s), 6.60 (1H, dd, J = 9.0,
1
3
2
.4 Hz), 7.32–7.35 (3H, m), 8.45 (1H, s), 9.67 (1H, s), 10.75 (1H, s); C NMR (CDCl , 600
3
MHz), δ (ppm): 12.4, 45.0, 56.3, 60.8, 96.9, 105.3, 108.8, 109.8, 112.5, 130.6, 141.2, 151.7,
+
1
53.1, 156.8. ESI-MS m/z: Calcd for C H N O (M+Na) : 476.1798, found: 476.1813.
2
4
27
3
6
2
.3. Preparation of electrospun nanofiber (ES) films
The electrospun nanofibers were prepared as presented in our previous report [44].
Briefly, a mixture of dimethylacetamide (DMAc) (60 g), acetone (40 ml) and polyurethane (20
g) was stirred for 12 h at room temperature. After the further slow addition of FCBH (45 mg) to
this mixture at room temperature, the resulting mixture was stirred for 12 h for fabrication. The
resulting clear, homogenous solution was used for electrospinning the film. The blended solution
was injected into a metallic needle using syringe pumps (KD Scientific model 200, USA) at a
constant rate of 0.5 mL/h. The tip of the metallic needle was connected to a high-voltage power
supply (Chungpa EMT, CPS-40K03VIT, Korea) that was set at 15 kV during the ES process.
The ES nanofibers were collected on a sheet of aluminum foil.
3
. Results and discussion
The chemosensor behavior towards various metal ions (Zn , Pb , Al , Co , Cr , Cu ,
2
+
2+
3+
2+
3+
2+
3
+
2+
2+
2+
2+
Fe , Hg , Mg , Mn and Ni ) was investigated by mass spectrometry and UV-Vis,
fluorescence, and proton NMR spectroscopic measurements. Visual detection, absorbance and
emission studies were performed in DMSO/water (1:1, v/v) at room temperature.

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2
+
3
.1. Colorimetric recognition of Cu
The binding ability of FCBH to various metal ions was studied by visual examination of
the metal-induced color changes in DMSO/water solution. Upon the respective addition of
2
+
2+
3+
2+
3+
2+
3+
2+
2+
2+
2+
equimolar quantities of Zn , Pb , Al , Co , Cr , Cu , Fe , Hg , Mg , Mn and Ni to a
2
+
2
0 µM solution of the FCBH, it was found that only Cu induced a rapid color change. The
2
+
addition of Cu caused a visible color change from greenish-yellow to red, as well as green
fluorescence quenching (Fig. 1). The color change was attributed to metal coordination with the
donor atoms (N and O) in FCBH. No color change was observed upon the addition of the other
chosen common metal ions. This observation suggests that the FCBH probe can serve as a
2
+
naked-eye colorimetric sensor for Cu .
Fig. 1. Naked-eye and fluorescence images of FCBH (20 µM, DMSO/water, 1:1) upon addition
of various metal ions (1 equiv.).
3
.2. Absorption spectroscopy studies
The UV-Vis spectra for the FCBH probe in the presence of various metal ions were
recorded in DMSO/water (1:1, v/v). The absorption spectrum of FCBH (20 µM) in DMSO/water
1:1, v/v) showed a major strong band centered at 457 nm (Fig. 2), which corresponds to the n–
(

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2+
π* transition. Upon addition of 1 equiv. of Cu , the absorption band at 457 nm red-shifted to
5
17 (Δλ = 60 nm), which was attributed to the intramolecular charge transfer (ICT) transition
from the donor to the acceptor [45,46], resulting in a perceived color change from greenish-
yellow to red. Other metal ions tested did not induce significant absorption changes under the
above conditions. These results indicate that the FCBH receptor has a remarkable selectivity to
copper ion, which is in good agreement with the colorimetric results.
1.00
0.75
0.50
0.25
0.00
2
+
3+
3+
3+
2+
FCBH, Co , Al , Cr , Fe , Hg
,
2+
2+
2+
2+
2+
Mg , Pb , Mn , Ni , Zn
2+
Cu
350
400
450
500
550
600
Wavelength (nm)
Fig. 2. Absorption spectra of FCBH (20 µM) before and after addition of various metal ions (20
µM).
2
+
To elucidate the interactions between FCBH and Cu , UV-Vis titration spectra were
2+
recorded with incremental additions (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µM) of Cu . With

ACCEPTED MANUSCRIPT
2
+
increasing concentration of Cu (0–1 equiv.), the absorption peak of FCBH at 457 nm gradually
decreased, and a new peak appeared at 517 nm, no further change was observed above 20 µM
(Fig. 3), which may indicate the formation of a new species. This result indicated that the
2
+
hydrazide was involved in the coordination with Cu . The changes at 457 and 517 nm were
monitored, demonstrating that the absorbance ratios are linearly related to the concentration of
2
+
2+
Cu in the 0–20 µM range (Fig. 3, inset), suggesting a 1:1 binding stoichiometry for the M-Cu
interactions.
2
+
Fig. 3. Absorption spectra changes of FCBH (20 µM) after addition of gradual amounts of Cu .
2
+
Inset: Absorption at 457 and 517 nm versus the concentration of Cu added.
3
.3. Fluorescence emission spectroscopy studies

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The fluorescence properties of the FCBH receptor were investigated at 450 nm excitation
2
+
2+
3+
2+
3+
2+
3+
2+
2+
2+
upon the addition of 1 equiv. of Zn , Pb , Al , Co , Cr , Cu , Fe , Hg , Mg , Mn and
2
+
Ni (20 µM) under the same conditions as mentioned in the absorption study (Fig. 4). The free
ligand FCBH showed a strong fluorescence emission band at 521 nm with a high quantum yield
2+
(
Φ = 0.302). Upon addition of Cu , the fluorescence diminished almost completely, and the
fluorescence quantum yield changed to 0.018. This phenomenon could be explained by chelation
2+
enhanced fluorescence quenching (CHEQ). Upon addition of Cu to FCBH, a stable chelate
2
+
complex of Cu with FCBH possibly induces rigidity in the resulting complex, thereby
generating efficient chelation-enhanced fluorescence quenching [47]. However, other metal ions
did not induce any distinct fluorescence quenching.
1000
2+
3+
3+
3+
2+
FCBH, Co , Al , Cr , Fe , Hg
,
2+
2+
2+
2+
2+
Mg , Pb , Mn , Ni , Zn
800
600
400
200
0
2+
Cu
500
550
600
650
Wavelength (nm)
Fig. 4. Fluorescence spectra of FCBH (20 µM) before and after addition of various metal ions
20 µM).
(

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A quantitative investigation of the binding affinity of FCBH was also performed by a
fluorescence titration experiment with the gradual addition (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and
2
+
2+
2
0 µM) of Cu (Fig. 5). With an increas in the concentration of Cu , the fluorescence intensity
at 521 nm of the probe solution decreased accordingly, and the fluorescence was almost
2
+
quenched after the addition of 1 equiv. of Cu . The fluorescence intensity of FCBH at 521 nm
2
+
exhibits a good linear relationship with the concentration of Cu between 0–20 µM (Fig. 5,
2
+
inset), which also strongly supports the 1:1 binding stoichiometry of the FCBH-Cu complex.
Fig. 5. Fluorescence spectra changes of FCBH (20 µM) after addition of the gradual amounts of
2
+
2+
Cu . Inset: Fluorescence intensity at 521 nm versus the concentration of Cu added.
2
+
3
.4. Stoichiometry of the FCBH-Cu complex

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2
+
The stoichiometry of complexation of FCBH with Cu was studied using a Job plot
obtained from fluorescence measurements and mass spectrometry analysis. The Job plot revealed
2
+
that the maximum fluorescence quenching was observed when the mole fraction of Cu was
approximately 0.5, which indicates a 1:1 ligand to metal stoichiometric ratio (Fig. 6). These data
were further confirmed by mass analysis. ESI-MS data showed the formation of a 1:1 complex
2
+
between deprotonated FCBH and Cu (Fig. S1). The observed mass peak at m/z 515.1
2
+
+
corresponded to [FCBH +Cu −H] (calcd for C H CuN O , 515.1).
2
4
26
3
6
1000
800
600
400
200
0
0.0
0.2
0.4
0.6
0.8
1.0
Mole fraction of copper
Fig. 6. Job plot obtained from fluorescence spectral data, where the intensity of FCBH at λmax of
2
+
5
21 nm was plotted against the mole fraction of Cu .
1
3
.5. H NMR spectroscopy studies

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To gain a better understanding the sensing mechanism of FCBH towards copper ions, a
1
2+
H NMR titration experiment was carried out in DMSO-d at room temperature (Fig. 7). Cu is
6
2
+
a paramagnetic ion that affects the chemical shift (δ) of protons that are close to the Cu binding
2+
site [48]. Upon the addition of Cu to FCBH, almost all the aromatic protons shifted downfield
due to the metal ion reducing the electron density on the conjugated receptor system, while no
shifts were observed for the aliphatic protons. The most obvious change in chemical shift was
2
+
noticed for the amide N-H proton. As Cu ions were added, the N-H proton shifted downfield,
and the signal gradually weakened, and then disappeared. The chemical shift signals of the
aromatic ring became very broad, weakened and almost completely disappeared upon the
2
+
2+
addition of Cu . These observations indicate that Cu binds to the FCBH chemosensor through
the N atom of the imine and the O atoms of C=O, which are directly connected to the aromatic
rings.

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1
Fig. 7. H NMR (300 MHz) spectral changes of FCBH on the addition of Cu(NO ) in DMSO-
3
2
d6).
3
.6. Theoretical (DFT) studies
To better understand their geometry and electronic structure, we carried out DFT
2
+
calculations for FCBH and FCBH-Cu . As shown in Scheme 2, the free receptor was found to
adopt a slightly twisted conformation. The geometry converts to planar upon complexation with
2
+
Cu . The spatial distributions and orbital energies of the highest occupied molecular orbital
2
+
(
HOMO) and the lowest unoccupied molecular orbital (LUMO) of FCBH and FCBH-Cu were
also generated using the calculations, as shown in Fig. 8. From the figure, it can be seen that the
electron density is mainly delocalized over coumarin hydrazone in the HOMO of FCBH, but in

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the HOMO of its copper complex, the electron density is located around the metal. The electron
density of the LUMO is extended over the whole molecule (mainly coumarin and
benzohydrazide) in FCBH, whereas in its copper complex it is mainly delocalized over
benzohydrazide. The HOMO-LUMO energy gaps of the free receptor and its corresponding
copper complex were found to be 3.284 and 2.159 eV, respectively. The decrease in the ΔE upon
2
+
2+
complexation with Cu corresponds to an ICT transition because the complex of FCBH-Cu is
a relatively better electron donor than the free receptor. The results demonstrated that the metal
complexation of the receptor lowered the HOMO-LUMO energy gap, and the reduction in the
energy gap can explain the bathochromic shift in the absorption spectra. Thus, the results
obtained from theoretical calculations were in good agreement with the experimental results.
2
+
Scheme 2. DFT-optimized geometries of FCBH and its corresponding Cu complex.

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2
+
Fig. 8. HOMO-LUMO energy level diagram of FCBH and its corresponding Cu complex.
3
.7. pH effect on the sensing behavior
The effects of pH on the fluorescence emission of FCBH in the absence and presence of
2
+
Cu (1 equiv.) were determined (Fig. 9). For FCBH, an obvious increase in the peak bonded at
21 nm was observed when the pH value changed from 2 to 7. A possible reason could be that
the highly protonated receptor is slowly dissociated into its free receptor. Similar spectral
5
2
+
changes were obtained for FCBH-Cu at acidic pH conditions, indicating that the protonation of
the receptor lowers its coordination ability, and a sudden decrease in emission at neutral pH is

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due to the formation of the metal complex. Over the pH range of 8–10, the fluorescence
quenching was observed in both cases. This can be explained by the fact that the deprotonation
occurs in alkaline pH conditions, which prevents the coordination ability of the receptor towards
the metal ion. The results reveal that the deprotonation process involved in the formation of the
2
+
2+
FCBH-Cu complex and the Cu recognition process is free from pH interference under neutral
pH conditions.
2
+
Fig. 9. Fluorescence intensity (at λmax of 521 nm) of FCBH and FCBH-Cu at various ranges of
pH.
3
+
3
.8. Competitive selectivity of CS to Al over other metal ions

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2
+
To further estimate the selectivity of FCBH for Cu , the competitive experiment in the
2
+
3+
3+
3+
2+
2+
2+
2+
2+
presence of other metal ions including Co , Al , Cr , Fe , Hg , Mg , Pb , Mn , Ni and
2
+
Zn under the same conditions was examined (Fig. 10). It is noticeable that upon addition of
2
+
Cu to FCBH in the presence of competitive metal ions shown immediate response in
absorbance and emission. Decrease in absorbance and quenching in emission could be due to
strong binding ability of copper ion and suitable coordination geometry conformation of ligand.
2
+
These results reveal that the recognition of Cu by probe FCBH is not significantly influenced
2
+
by other competitive metal ions and it could be used as a selective chemosensor for Cu .
Fig. 10. UV-Vis absorbance (at λmax = 457 nm) and fluorescence emission (at λmax = 521 nm)
2
+
responses of FCBH to Cu in the presence of various metal ions.
3
.8. Practical applications
FCBH is a favorable selective colorimetric and fluorescent chemosensor for Cu in
DMSO/water (1:1, v/v) media. To get a practical application view, electrospun nanofibers
2
+
2
+
blended with FCBH were prepared to sense Cu in aqueous solution. As we expected, receptor-
blended ES test strips were successfully examined in water/EtOH (9:1, v/v).

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The morphologies of the electrospun nanofibers were characterized by SEM analysis.
The film was composed of numerous, smooth and randomly oriented cross-linked nanofibers
(Fig. S2). FCBH-blended nanofibers exhibited similar morphology to that observed in pure
polyurethane nanofibers. The SEM results showing the uniform distribution of material on the
nanofibers. However an electrospun fiber from this process typically has a diameter in the range
of 500-1000 nm. We obtained small nanofibers in large quantities and with a uniform size.
2
+
FCBH-blended ES thin films were further used as test strips for the detection of Cu .
To examine selectivity, the test strips were sprayed with solutions containing various
2
+
2+
3+
2+
3+
2+
3+
2+
2+
2+
2+
cations, such as Zn , Pb , Al , Co , Cr , Cu , Fe , Hg , Mg , Mn and Ni (100 µM). As
shown in Fig. 11, a significant visible color change from greenish-yellow to red was observed
2
+
immediately after the addition of Cu , and green fluorescence quenching was also observed
under UV light. Furthermore, the sensing behavior of the FCBH-blended electrospun thin film
was also investigated by confocal laser scanning microscopy (CLSM). A mixture of ethanol and
2
+
water (9:1, v/v) containing Cu (100 µM) was dropped onto the surface of an FCBH-blended
nanofiber strip, and CLSM was subsequently performed using an excitation wavelength at 475
nm. The free state of the FCBH-blended nanofiber shows a bright yellow fluorescence (Fig. 12).
The fluorescence on the surface of the nanofibers is almost completely quenched upon addition
2
+
of Cu . This experiment indicates that the sensor is quite useful for the rapid in situ detection of
2
+
Cu .

ACCEPTED MANUSCRIPT
Fig. 11. Color change of FCBH-blended electrospun nanofiber test strips after treatment with
various metal ions under normal (a) and UV (b) lights.
Fig. 12. Fluorescence confocal images of color variations of FCBH-blended electrospun
2+
nanofibers upon treating with Cu ions.
2
+
3
.9. UV-Vis and fluorescence response of the FCBH-Cu complex towards anions
We subsequently studied the chemical reversibility behavior of FCBH-Cu by
absorption and fluorescence experiments in DMSO-water solution. For this purpose, FCBH-
2
+
2
+
−
−
−
−
−
−
−
−
−
Cu was treated with a variety of anions such as Cl , Br , I , F , OH , CN , SCN , HS , N ,
3

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−
−
−
4
NO , ClO and H PO (20 µM). As depicted in Fig. 13, UV-Vis spectra returned to their
3
4
2
2
+
original state of FCBH, and the quenched fluorescence of the FCBH-Cu complex was restored
−
2+
upon addition of CN to the solution of FCBH-Cu . Meanwhile, the red color of the FCBH-
2
+
blended electrospun thin film with Cu immediately returned to the initial yellow color upon the
−
addition of CN (Fig. 13, inset), resulting from the formation of more stable copper complexes
3
such as [Cu(CN) ] ⁻ and [Cu(CN) ]⁻ [49]. The color of the recovered FCBH-blended
4
2
electrospun nanofiber thin film was regenerated upon further addition of copper ions. However,
other anion species caused almost no change in the absorption and fluorescence spectra under the
same conditions.
2
+
Fig. 13. Absorption (a) and fluorescence (b) spectra of FCBH-Cu in the presence of various
anion specious. Inset: Images of proof of reversibility.
The reversibility and regeneration of sensing probes are important factors in practical
2+
−
applications. The alternate additions of a constant level (20 µM) of Cu and CN to the solution
of FCBH gives rise to a switchable change in the fluorescence emission at 521 nm (Fig. 14).

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This reversible fluorescence behavior of FCBH can be repeated several times by alternating
2
+
−
Cu /CN addition. These results demonstrate that the metal binding of FCBH is chemically
reversible, and the FCBH probe can be used as a reversible colorimetric and fluorometric
2
+
chemosensor for Cu ions.
1000
800
600
400
200
0
-
-
-
-
-
CN
CN
CN
CN
CN
Cu
Cu
Cu
Cu
Cu
2+
2+
2+
2+
2+
2+
FCBH+Cu
0
1
2
3
4
5
Cycles
Fig. 14. Changes in fluorescence of FCBH (20 µM) at 521 nm upon alternate addition of a
2
+
−
constant amount of Cu and CN (1 equiv.).
2
+
From previous studies, the possible binding mode between FCBH and Cu is shown in
Fig. 15. The fluorescent ligand FCBH is a slightly twisted, non-planar, highly conjugated π-
2
+
electron system. Upon addition of Cu , the non-planar structure is converted into a planar

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structure, and the fluorescence of the ligand is completely quenched due to the strongly bonded
2
+
paramagnetic nature of Cu ion.
2
+
Fig. 15. Proposed mode of complexation between FCBH and Cu . Observed naked-eye (left)
2
+
and fluorescence (right) changes upon FCBH-Cu complexation and subsequent treatment with
−
CN are shown.
4
. Conclusions
In conclusion, we have synthesized a new coumarin-based chemosensor (FCBH), which
2
+
showed a reversible fluorescence quenching response toward Cu via a 1:1 binding mode in
2+
aqueous solution. The addition of Cu causes a visible color change from greenish-yellow to
red, as well as green fluorescence quenching. Meanwhile, the FCBH probe displayed excellent

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2
+
selectivity for Cu over other tested metal ions. The binding mode and the corresponding
1
quenching mechanism were examined by UV-Vis, fluorescence, ESI-MS, and H NMR
2
+
techniques, as well as DFT calculations. The Cu -induced fluorescence quenching can be
attributed to CHEQ. Furthermore, FCBH-blended electrospun nanofiber test strips could
2
+
2+
selectively detect Cu ions in aqueous solution. Further studies of the FCBH-Cu mixture
−
revealed that the fluorescence quenching was reversible in the presence of CN ions.
Acknowledgements
This study was supported by the National Research Foundation of Korea (NRF) funded by the
Ministry of Science, ICT and Future Planning (Grant no. NRF-2014M3C1A9060739).
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