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 Page 1 of 9 RSC Advances
View Article Online
DOI: 10.1039/C6RA03021A

A Colorimetric and Fluorometric Investigation of Cu(II) ion in

Aqueous Medium with a Fluorescein-based Chemosensor
Published on 23 March 2016. Downloaded by University of California - Santa Barbara on 23/03/2016 11:23:24.

Reena Rathod, Smritilekha Bera, Man Singh, and Dhananjoy Mondal*

School of Chemical Sciences, Central University of Gujarat, Gandhinagar- 382030, India

RSC Advances Accepted Manuscript

Fax: +91-79-23260076; Tel.: +91-79-23260210

KEYWORDS: Copper(II) ion, Fluorescein, Schiff base, UV-Visible, colorimetric, fluorescence spectroscopy

ABSTRACT:

As a selective colorimetric and fluorometric chemosensor, a novel fluorescein-based Schiff base (FNSB), 1,4-bis(1-
fluorescein)-2,3-diaza-1,3-butadiene (L, 3) exhibits efficient binding for Cu2+ ion in water and allows naked-eye detection
as a result of the formation of a 1:1 copper-ligand complex. The complexations of Cu2+ ion due to molecular interaction
with 3 resulted in the rapid change in color from colorless to deep yellow with a characteristic change in UV-Vis absorp-
tion to a longer wavelength by 130 nm in acetonitrile and a significant quenching of the fluorescence intensity (∼2.5 fold
for Cu2+ ion) with maxima at 519 nm in 1% DMSO-Tris buffer solution at physiological pH 7.4. The detection limit of Cu2+
ion in acetonitrile is found to be 20 µM in UV spectroscopy whereas in aqueous solution the limit is reduced to 1.25 µM in
fluorescence spectroscopy.

Metal ions allied environmental pollutants and their tox-
icity are globally focused problems in recent times due to
their critical role in physiology, ecology, evolutionary,
nutrition, and environment.1 In this respect, there is an
immense need for detection and identification of metal
ions in aqueous solution for environmental monitoring
and biomedical science, ensuring the regular functionality
of ecosystem, health and humanity. It is found that, the
involvement of metal ions in a specific concentration in a
number of biological processes is essential.2 In particular,
the copper plays a pivotal role in different biological pro-
cesses such as iron absorption, haemopoiesis, various en-
zyme-catalyzed and redox reactions.3 Although an aver-
age neural copper concentrations in order of 0.1 mM is
required in the body and brain for biological processes
but the higher concentration causes vomiting, lethargy,
increased blood pressure and respiratory rates, acute he-
molytic anemia, liver damage, neurotoxicity, and neuro-
degenerative diseases.4,5 Furthermore, copper ions can
disrupt natural ecosystems due to their adverse effects on
microorganisms.6 Thus, there is an upsurge of interest in
the development of selective and reliable detection meth-
ods for the qualitative and quantitative analysis of metal
ions. In this perspective, chemosensors are of great im-
portance to chemistry, biology, and medicine because
they allow rapid detection of different compounds in the
living organisms and environment.2
 RSC Advances
Of particular interest, colorimetric and fluorometric
chemosensors find distinct advantages in terms of sensi-
tivity, selectivity, response time, real time analysis and
easy operating system. To date, several molecular design
strategies have been exploited to develop chemosensors
for selective recognition of different species on the basis
of different host-guest interactions, such as hydrogen-
bonding, electrostatic force, metal-receptor coordination,
hydrophobic and van der Waals forces of interactions7 but
Published on 23 March 2016. Downloaded by University of California - Santa Barbara on 23/03/2016 11:23:24.
the effective sensors in context of compound selectivity,
solvent specificity and detection limit are inadequate.
In recent time, several fluorophores based on boron-
dipyrromethene (BODIPY), naphthalene, pyrene, anthra-
quinone, coumarin, xanthane type, rhodamine and fluo-
rescein, which are effective either in UV or Vis/near IR
region, have been studied.8 Among them fluorescent
chemosensors for Cu(II)-selective detection were reported
and have been used with some success in biological appli-
cations.9 However, lacking of poor water solubility and
selectivity makes the sensors unsuitable for environmen-
tal and cellular applications. To address these limitations,
water soluble fluorescein-based chemosensor is signifi-
cant due to its intrinsic advantages of high quantum
yield, absorption and emission maxima in visible region
over other organic dyes.10,11
In addition to the signaling unit of fluorophore with
built-in Lewis base receptor i.e, Schiff base receptors with
N-donor atoms of the imine group and O-donor of the
phenol as a recognition part usually coordinate to a cation
as Lewis acid affording stable complexes with various
transition metals.12a-e Thus, the presence of amines/imines
in the receptor plays a key role of establishing electronic
communication between the fluorophore and analytes.12f-g
Many methods have been used to detect metal ions in-
cluding fluorescence spectroscopy, UV-Vis absorption,
atomic absorption, ICP emission spectroscopy, and volt-
ammetry. Amongst these, UV-Vis absorption and fluores-
cence emission spectroscopy are attractive approaches
because of their high sensitivity, facile operation, and the
widespread availability of equipment for analysis. As a
result, fluorescent chemosensor molecules have been de-
signed and synthesized for metal ion detection in some
cases yielding an enhanced (turn-on) signals and
quenched (turn-off) fluorescence signals13,14 at low con-
centrations to develop fluorescence turn-on/off sensors.
In this contribution, we report a new assay for the de-
tection of Cu2+ ion using fluorescein-based Schiff base
(FNSB), 1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene (3)
as a highly selective colorimetric and fluorometric
chemosensor in organic solvent as well as in aqueous so-
lution at physiological pH = 7.4, respectively. Indeed, the
inherent ability15 of Cu2+ ion to quench fluorescence over
other quenching metal ions (Hg2+, Ni2+, Pb2+, and Ag+) has
been exploited to develop the selective fluorescence
quenching sensor.
Though ample numbers of ligands are reported in liter-
ature for selective detection of Cu2+ in organic solvents9
and a few are found to be used in aqueous medium9v but
Page 2 of 9
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DOI: 10.1039/C6RA03021A
ligands are rarely found active both in organic and aque-
ous medium9w. Fortunately, our synthesized imine-
functionalized fluorescein-based ligand 3, which is ob-
tained in two-step reaction from the naturally occurring
inexpensive fluorescein with moderate to good yield, se-
lectively binds Cu2+ in both organic and aqueous media
without affecting the specificity or selectivity to other
cations.
Several factors such as high quantum yield, absorption
and emission maxima of fluorescein and the participation
of Schiff base as a recognition unit for coordination to a
metal cation as Lewis acid were considered in the design
of fluorescein-derived Schiff base ligand. In addition to
the above parameters, the pH as exogenous factor affect-
RSC Advances Accepted Manuscript
ed the chromogenic behavior (or selectivity and sensitivi-
ty) of receptor. Generally, fluorescein exists in a fluores-
cent ring opened carboxylic acid form and in a non-
fluorescent ring closed lactone form and this equilibrium
is very sensitive to the pH of the solution (Figure 1).16
Thus, this pH-tunable sensitivity of fluorescein can be
used to sense changes in a local environment by monitor-
ing its ring opening and closing equilibrium in addition to
other spectroscopic effect to detect the metal ions from
industrial and commercial pollutants.17 In this regard, the
development of water-soluble fluorescein-derived Schiff
base fluorophore for the tunable detection of Cu2+ ion in
the aqueous solution at physiological pH is an attractive
and challenging approach to be addressed.
EXPERIMENTAL SECTION
General Information and Methods. All materials were
obtained from commercial suppliers and were used with-
out further purification. All the solvents were dried ac-
cording to the standard methods prior to use. All of the
solvents were either HPLC or spectroscopic grade in the
optical spectroscopic studies, and all reactions were mon-
itored by TLC analyses on aluminium plates pre-coated
with silica gel 60 F254. Column chromatographic purifica-
tions were carried out on silica gel (200-400 Mesh size).
NMR spectra were measured on a Brukar Avance. The 1H
NMR (500 MHz) chemical shifts were given in ppm rela-
tive to the internal reference TMS. The 13C NMR (125
MHz) chemical shifts were given using CDCl3, MeOH-d4
and DMSO-d6 as the internal standard. ESI-MS data were
recorded on a Agilent Q-TOF MicroTM LC-MS instru-
ment mass spectrometer. Fluorescence excitation and
emission spectra were obtained using Edinburgh instru-
ment Xenon-900 Spectrofluoro photometer. UV−vis ab-
sorption spectra were recorded on a spectro2060+
UV−visible spectrophotometer.
Synthesis of 1,4-bis(1-fluorescein)-2,3-diaza-1,3-
butadiene (3). Scheme 1 illustrates the two step synthesis
of fluorescein-derived Schiff base (FNSB), 1,4-bis(1-
fluorescein)-2,3-diaza-1,3-butadiene (3) as cationic ligand
(L). The Reimer-Tiemann reaction of fluorescein (1) with
chloroform in alkaline aqueous methanolic solution at 55
ºC produced fluorescein monoaldehyde (2)18 in 24-28%
yield. The appearance of peak at 10.63 ppm in the 1H NMR
spectrum (Figure SI 1) and C-H stretching of –CHO due
to Fermi coupling at 2928, 2853 cm-1 and C=O stretching
 Page 3 of 9 RSC Advances
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DOI: 10.1039/C6RA03021A
-1
at 1727 cm in FTIR (Figure SI 6) confirmed the func- plex. The melting point of the ligand was also determined
tionalization of fluorescein with aldehyde at Reimer Tie- and it is found to be 350 oC.
mann reaction. The Schiff base formation was performed O
by the reaction between fluorescein monoaldehyde and O CO2H CO 2H
f luorophore
slightly excess of hydrazine hydrate in ethanol at 90 °C
affording the final 1,4-bis(fluorescein)-2,3-diaza-1,3- HO
HC
O
spacer
OH O
1% DMSO-Tris buf fer
O OH O
Cu(ClO 4)2
O O
HC CH
butadiene (3) in 35% yield. The appearance of mass peak N
N
receptor
pH = 7.4 N
N
1% DMSO-Tris buff er
pH = 7.4
HN
Cu2+

at m/z = 717.1558 for [M+H]+ in the mass spectroscopy CH spacer pH = 4 CH N

HO O OH O O OH CH
O O O
(Figure SI 5), the disappearance of aldehyde peak at 10.63
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f luorophore
ppm in the 1H NMR spectra, C-H stretching at 2928, 2853 O HO 2C
HO 2C

cmˉ1 and C=O stretching at 1727 cmˉ1 in FTIR spectra

O
fluorescent (L)
1,4-bis(1-fluorescein)-2,3-diaza-1,3-butadiene
(Figure SI 6) indicate the formation of Schiff base 3 from non-fluorescent
Cu-L
non-fluorescent

aldehyde 2 with hydrazine hydrate (Scheme 1).

Scheme 2. Proposed mechanism of the sensoring system
Scheme 1. Synthesis of 1,4-bis(1-fluorescein)-2,3-diaza-

RSC Advances Accepted Manuscript

1,3-butadiene (3) as cationic ligand The details of procedure and spectral data of the com-
pounds could be found in Supporting Information.
H O

HO O OH HO O OH
NaOH, MeOH, CHCl3
O O
RESULT AND DISCUSSION
55 °C, 12h

O O Solubility Studies. Investigation of selectivity of the syn-

1
fluorescein O
2
fluorescein monoaldehyde thesized cationic chemosensor 3 towards different metal
O
fluorophore (F)
ions necessitates the choice of organic solvent, aqueous
buffer and overall solvent composition. The solubility
HO O OH
HC
study of the cationic chemosensor (3) was performed with
N
N 2 H4 .H 2 O, ethanol N
CH UV-Vis spectroscopy at concentration of 2.5×10-5 M in
HO O OH
reflux, 22 h
different organic solvents and aqueous medium. The out-
O
fluorophore come revealed that the solubility in organic solvent with
O polarity index ranging from 5.1-7.2 (DMF, DMSO, pyri-
3 (L)
1,4-bis(1-flurescein)-2,3-diaza-1,3-butadiene dine, acetone, acetonitrile) is noticeable with highest in-
no fluorescence
tense broad peak at 350 nm. The synthesized ligand was
non-fluorescent and solubility in aqueous solution (pH<
Spectral Studies. The selectivity, binding and stoichiome- ~7.0) is not noteworthy. But the compound 3 is highly
try of the complex system were also determined by the soluble in 1% DMSO in aqueous Tris-buffer at pH = 7.4
NMR and Mass spectroscopy. NMR and Mass spectrosco- appearing as fluorescent with absorption peak at near 491
py data support 1:1 ligand-to-metal complex formation nm. The solubility of the sensor in different organic sol-
(Figure SI 7 and SI 8) with change of binding constant in vent cropped up with the visibility of color change in na-
fluorogenic response of ligand 3 in the presence of varying ked-eye and these phenomena are also supported by the
concentrations of Cu2+ indicating the formation of a 1:1 UV-Vis spectroscopy study. In this regard, it is to be men-
ligand-to-metal complex (by Job’s plot (Figure 13), NMR, tioned that though the ligand was highly soluble in DMF
MASS) with an association constant of 2.23×104 M-1 (Fig- and DMSO with absorption peak at near 350 nm but their
ure 12). The conversion of L to L-Cu2+ i.e (L:Cu2+ in 1:1) intrinsic coordinating, oxidizing property, and high boil-
(Scheme 2) was confirmed by ESI-TOF-MS and 1H NMR ing point caused the limitation of use as the experimental
spectrum. The peak at m/z 779.20 in positive ESI mode medium. Thus, comparably good solubility of the ligand 3
and 778.32 in negative ESI mode corresponding to mass of in acetonitrile at 2.5×10-5 M concentration with absorb-
[L-Cu2+] (calcd m/z 779.07) was found after 1 equiv. of ance peak at 350 nm favoring the detection of metal ions
CuClO4 was added to the solution of 1 equiv. ligand (Fig- in naked-eye by the measurement of peak shifting by
ure SI 8). The cation binding by ligand 3 was also detected spectroscopic study (Figure 1).
by changes in the 1H NMR spectrum. In the 1H NMR spec-
trum, upon addition of variable amounts of Cu2+, the
shifting of the ligand protons and the change of splitting
of the ligand protons showed the formation of ligand-to-
Cu2+ complex. Moreover, after the cation concentration
has reached the ligand concentration, further addition of
Cu2+ is ineffective. Above all, the TLC (Thin Layer Chro-
matography) also clearly shows that the ligand-
Cu2+complex is formed by the Rf value 0.0 in comparison
to the Rf = 0.50 of free ligand in 1:20 MeOH/DCM mixture
(Figure SI 9). Thus, ESI-TOF-MS, NMR and TLC results
firmly support the inter-conversion of L to L-Cu2+ com-
 RSC Advances Page 4 of 9
View Article Online
DOI: 10.1039/C6RA03021A
1.6 MeOH ons with the ligand 3 in 10:1 stoichiometric ratio. In this
ethanol
1.4
water
scenario, all metal ions in acetonitrile exhibit instant col-
1.2 chloroform or change detected by UV spectra. While the ratio of met-
DMF
1
al and ligand 3 was changed to 2:1 (Figure 3a), the selec-
pyridine
absorbance

DCM tive detection of Cu2+ and Hg2+ cations was witnessed with
0.8
1% DMSO-TRIS (pH = 7.4) a new band formation at 480 nm and 440 nm, respective-
0.6 DMSO
acetone
ly. This result demonstrates that Cu2+ ion only gives max-
0.4 ACN imum quantifiable change with red shift of 130 nm along
with change in colour from light pink to yellow. But the
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0.2

0
interesting fact is that the absorbance intensity of Cu2+
300 350 400 450 500 550 600 and Hg2+ cation decreases whereas the intensity of Ni2+
wavelength cation increases with time and solution became pink col-
or (Figure 3b).

RSC Advances Accepted Manuscript

Figure 1. UV-Vis absorbance of ligand 3 in various solvents 1
L
-5
at 2.5×10 M concentration 0.9 Cu2+

0.8 Hg2+
pH Studies. To inspect the pH effect and the solubility of 3
0.7 Li+
in aqueous solution, the absorbance of chemosensor at con- 2+

absorbance
-5 Cu Na+
centration 2.5×10 M was investigated in the pH range of 2-10 0.6
K+
in 1% DMSO-Tris buffer solution. It is found that the ligand 3 0.5
shows no solubility in aqueous medium at acidic and even in Mg2+
0.4
neutral pH. The absorbance of 3 in basic media ranging from Hg
2+ Ca2+

7.4 to10.0 pH in Tris base at the concentration of 1 mmol and 0.3 Ni2+
10 mmol exhibits similar fluorescent activity with same ab- 0.2 Zn2+
sorbance maxima at 491 nm but with difference in intensities 0.1 Ag+
(Figure SI 11). The result in acidic or neutral pH is not signifi-
0
cant presumably due to the stability of ring closure form of 250 300 350 400 450 500 550 600
fluorescein as non-fluorescent. The wavelength (nm)

Figure 2. pKa-dependent structure of fluorescein

changes in pH value from 7 to 7.4 and addition of 1%
DMSO in aqueous Tris buffer solution, which is compati-
ble in biological system, show very good absorbance
property of the synthetic ligand 3 at higher wavelength of
ca. 141 nm in comparison to organic solvents due to the
extended conjugation of the phenolate anion at pH 7.4
(Figure 2).
UV-Vis spectral Studies of the metal-ligand complex.
The metal-binding properties of the ligand 3 in organic as
well as in aqueous solution is examined. Colorimetric
study of metal-ligand interaction was performed in ace-
tonitrile to determine the selectivity and sensitivity of
these metal cations (Figure 3): Li+, Na+, K+, Mg2+, Ca2+,
Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, Pb2+, Pr3+, Sm3+, and Gd3+.
The study commenced with nitrate salt of lanthanide
metals and perchlorate salt of the remaining metal cati-
Figure 3. (a) UV-Vis spectra of the fresh solution of ligand 3
-5 -
at 2.5×10 M in acetonitrile in presence of metal ions at 5×10
5
M (b) UV-Vis spectra of the same solution of ligand 3 and
2+
Ni after 6 h.
Studies of sensitivity of the ligand. To determine the
limit of detection (LOD) for the Cu2+ cation in acetoni-
trile, different concentration of Cu2+ ion (10 to100 ppm)
was titrated against ligand 3 at 2.5×10-5 M concentration
and detected by UV-Vis spectroscopy. The LOD for the
Cu2+ ion from absorbance spectra was found to be 20 µM.
Surprisingly, after 1h, metal-ligand solution started to
change their corresponding absorbance, which concluded
that the complex formed during metal-ligand binding
study in acetonitrile is not stable in the solution for long
time.
 Page 5 of 9 RSC Advances
View Article Online
DOI: 10.1039/C6RA03021A
0.9 L

0.8 100 µM
50 µM
0.7
40 µM
0.6
absorbance
35 µM
0.5 L
30 µM 7.00E+05
0.4 Cu2+
25 µM Na+
0.3 6.00E+05 Hg2+
20 µM

fluorescence intensity
0.2 Ag+
10 µM 5.00E+05 Cd2+
0.1 K+
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0 Pb2+
4.00E+05
310 360 410 460 510 560 Li+
wavelength (nm) Ni2+
3.00E+05 Ca2+
Zn2+
2+
Figure 4. Titration curve of Cu against ligand 3 at concen- 2.00E+05 Mg2+
-5 Pr3+
tration 2.5×10 M in acetonitrile Gd3+

RSC Advances Accepted Manuscript

1.00E+05 Sm3+
After colorimetric study of ligand 3 in organic solvent, the
0.00E+00
lower detection limit of synthesized ligand with Cu2+ ion 490 540 590 640
as fluorescent probe at physiological pH 7.4 was also de- wavelength
termined through colorimetric methods. To execute this
experiment, spectroscopic measurements of ligand 3 are L
investigated in 1% DMSO-Tris buffer at physiological pH 0.4 Cu2+
Na+
(10 mM Tris buffer, pH 7.4) under ambient condition. Hg2+
0.35
Addition of biologically and environmentally relevant Ag+
0.3 Cd2+
alkali, alkaline earth, transition, lanthanides or heavy

absorbance
K+
metal ions did not produce any discernible change in the 0.25 Pb2+
Li+
absorption spectral profile of the receptor at lower con- 0.2 Ni2+
centration of metal ions (~10-5 M). This observation sug- Ca2+
0.15 Zn2+
gests no interaction between the synthesized chemosen- Mg2+
sor and the added metal ions. However, in the presence of 0.1 Pr3+
Cu2+ ion, prominent changes in the UV-Vis spectra are 0.05
Gd3+
Sm3+
observed. The metal-free receptor gives strong fluores-
0
cence emission (I = 672420) upon excitation at 491 nm 400 450 500 550 600
due to high affinity with chemosensor 3 through efficient wavelength
photoinduced electron transfer (PET) from the fluoro-
-
phore to the receptor at physiological pH 7.4. This emis- Figure 5. (a) Fluorescence spectra of chemosensor 3 at 1.5×10
5
sion profile does not change to any noticeable extent in M in 1% DMSO-Tris buffer (10 mmol) at pH 7.4 in presence
-4
terms of both intensity and peak positions in the presence of metal ions (1.5×10 M) at λex = 491 nm and slit with 2 nm.
-5
of alkali, alkaline earth, transition, or lanthanide metal (b) UV-Vis absorption spectra of 3 at 1.5×10 M in presence of
-4
ions. While the addition of the perchlorate salt of Cu2+ metal ions (1.5×10 M) in 1% DMSO-Tris buffer at pH 7.4.
elicits a significant diminution of the fluorescence inten-
sity (∼2.5 fold for Cu2+ ion) with a wavelength of maxi- When the experiment was performed in fluorescence
mum absorption at 519 nm for Cu2+ (Figure 5a). The turn- spectrophotometer, metal ions concentration is 10 times
off fluorescence response is found to be reversible as addi- to the ligand (3), an excellent selectivity was observed
tion of an aqueous solution of ligand to the L-Cu2+ solu- towards Cu2+ ion with fluorescence quenching among all
tion changes the fluorescence output to the level of met- other metal cations and the quenching intensity was ob-
al-free receptor; this observation was also supported by served from 6.7×105 to 2.9×105. But a significant fluores-
the Job’s plot. cence change was not noticed in fluorescence spectrosco-
py for other metal cations. This phenomenon unambigu-
With ligand concentration at 15 μM with 10 times more ously indicate that the binding interaction between the
metal ion concentration shows the change in absorbance ligand 3 and Cu2+ is stronger than the interaction between
peak in the UV spectrophotometry from 490 to 496 nm the ligand 3 and other quenching metal ions (Figure 6).
with an intense absorbance peak wherein the ligand 3
shows absorption peak at 496 nm (Figure SI 12). This
small change in absorbance makes difficulty in the selec-
tivity of metal cation (Figure 5b). The association of the
receptor with a complementary analyte can suppress ei-
ther an electron or an energy transfer process and switch
on the fluorescence of the adjacent fluorophore.
 RSC Advances Page 6 of 9
View Article Online
DOI: 10.1039/C6RA03021A

fluorescence intensity
800000
y = 3587.3x + 34866
700000 2.0E+5
R² = 0.9604
600000
1.8E+5
500000
1.5E+5
400000

300000 1.3E+5

I0 - I
200000
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1.0E+5
100000
7.5E+4
0

5.0E+4

metal ions
2.5E+4

RSC Advances Accepted Manuscript

-5
Figure 6. (a) Fluorescence intensity of ligand 3 at 1.5×10 M 0.0E+0
in 1% DMSO-Tris buffer (pH 7.4) in presence of various metal 0 5 10 15 20 25 30 35 40 45 50
-4
ions (1.5×10 M) at λmax = 519 nm. Cu2+ in µM

Studies of Fluorometric titration of Cu2+ against lig- Figure 8. LOD curve for Cu against ligand concentration at
2+

and. For the limit of detection (LOD) at pH 7.4, the titra- 5.0 µM in 1% DMSO-Tris base at pH 7.4.
tion curve of Cu2+ was figured out by considering concen-
tration of metal ions from 0.2 to 100 equivalents with re- Sensing Mechanism Studies. The addition of acid main-
spect to the ligand 3 in 1% DMSO-Tris buffer (Figure 7). tains the lactone form of chemosensor, activating an en-
Surprisingly, the detection limit was found to be 1.25 µM ergy transfer pathway and quenching effectively the fluo-
in aq. solution (Figure 8) in fluorescence spectroscopy rescence of the receptor.19 The addition of base converts
while according to UV spectroscopy, the detection limit the chemosensor into the carboxylic acid form (Figure 9),
of Cu2+ cation was found to be 20.0 µM in acetonitrile preventing the electron transfer process and switching on
(organic solvent). Hence, the detection limits via fluores- the fluorescence of the receptor.
cence spectroscopy provides sixteen-fold increase in receptor receptor
aqueous medium than UV spectroscopy in organic sol-
vent. analyte analyte
f lurophore flurophore

L energy transf er
450000 1µM
electron transfer (A)

1.25µM
400000 2.5µM
5µM energy transf er energy transfer
10µM receptor
receptor receptor receptor
350000 15µM
20µM H+ OH-
fluorescence intensity

25µM analyte
300000 30µM
35µM f lurophore
f lurophore flurophore f lurophore
40µM
250000 45µM electron transfer electron transf er
50µM (B)
100µM
200000 150µM
200µM Figure 9. Molecular interactions
150000 250µM
300µM

100000
350µM
400µM The mechanism of Cu2+-mediated fluorescence
425µM
450µM
quenching in chemosensors: Non-radiative deactiva-
50000 475µM
500µM
tion of a photoexcited fluorophore 3 by the interaction of
Cu2+ ion can be achieved either through “energy transfer”
0
500 520 540 560 580 600 620 640 or “electron transfer”.20 Since, transition metal ions like
wavelength (nm) Cu(II) possess empty or half-filled orbitals, which can be
involved in an Dexter type energy-transfer mechanism as
Figure 7. Fluorometric titration of Cu2+ against ligand per the presentation in Figure 10A. On the other hand,
concentration at 5.0 µM in 1% DMSO-Tris base at pH 7.4. the coordination of N-atom of the Schiff base 3 favors an
access to the Cu(III) state21 in the Dexter type electron-
transfer mechanism. Thus, electron can transfer from the
complexed Cu(II) centre to the photoexcited fluorophore
moiety F* of 3, as depicted in Figure 10B.
 Page 7 of 9 RSC Advances
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DOI: 10.1039/C6RA03021A
A. energy transf er Figure 12. Determination of binding constant (association
2+
constant) to find binding affinity of ligand for Cu in 1%
x2-y2 x2-y2 DMSO-Tris buffer at pH 7.4; I0 is the fluorescence intensity
of L; I is the fluorescence intensity of L+[Cu2+] and Iα =
z2 minimum fluorescence intensity of L+[Cu2+].
z2

Cu(II) *Cu(II) Studies of Job’s Plot. The observed peak abscissa values
F* F
of 0.50 in Figure 13 indicate 1:1 stoichiometry of binding
(ligand 3/Cu2+) for ligand 3 (theoretical value is 0.50).
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B. electron transfer
Hence from the above study it is revealed the proposed
mechanism for the detection of Cu2+ as shown in Scheme
x2-y2 x2-y2
2.

z2 z2 2.5E+05

RSC Advances Accepted Manuscript

F *Cu(III) 2.0E+05
F* Cu(II)
1.5E+05

(I0-I)X
Figure 10. Quenching of the excited fluorescein-based Schiff
1.0E+05
base in presence of Cu(II) ion: A. Dexter type energy transfer
mechanism; B. Dexter type electron transfer mechanism. 5.0E+04

Determination of Stern–Volmer and Association con- 0.0E+00

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
stant. This unambiguous result is also corroborated from X
binding constant changes in the fluorogenic response of
ligand 3 in presence of varying concentrations of Cu2+ Figure 13. Job’s plot of Cu2+ complex formation with 3 at 5
with an Stern–Volmer constant of Ksv = 1.69×104 M-1 and µM concentration of each in 1% DMSO-Tris buffer at pH 7.4.
association constant Kass = 2.23×104 M-1 (Figure 12) which 2+
X = [3]/[3]+[Cu ] is the mole fraction of the 3, I0 is the fluo-
indicates the affinity of Cu2+ with the ligand via a static rescence intensity when X = 1 and I is the fluorescence inten-
quenching pathway. sity at respective values of X. Samples had 5 µM concentra-
2+
tion of each 3 and Cu at 519 nm.
2.1

1.9 y = 0.0169x + 1.0484

R² = 0.968 CONCLUSION
1.7
I0/I

1.5 In summary, we have synthesized a novel fluorescein-

1.3
based chemosensor 3 for selective binding to Cu2+ metal
cation in acetonitrile as organic solvent and in water (1%
1.1 DMSO-Tris buffer solution) at pH 7.4. According to UV
0.9
spectroscopy, the detection limit of Cu2+ was found to be
0 10 20 30 40 50 60 20 µM in acetonitrile whereas the detection limit in 1%
Cu2+ in µM DMSO-Tris buffer at pH 7.4 was reduced to 1.25 µM in
fluorescence spectroscopy. In this buffer solution at pH
Figure 11. Stern-Volmer plot for ligand 3 as a function of the 7.4, the receptor shows fluorescence quenching. This
2+
concentration of Cu cation sensing ability at physiological pH in aqueous media
might be useful for the detection of Cu2+ in vivo as copper
0.1 is a widely used industrial metal, and toxic at high con-
0.05 y = 1.1379x + 4.9535 centration and is involved in brain diseases like Alz-
log (I0-I/I-Iα)

0 R² = 0.9692 heimer’s, and Parkinson’s etc. Even though some colori-

metric/fluorescent chemosensors have been developed
-0.05
for the detection of Cu2+ so far, the sensing methods for
-0.1
fast detection of Cu2+ in aqueous solution, especially using
-0.15 colorimetric sensors without resorting to instruments, are
-0.2 relatively rare. This fluorescent dye works in aqueous me-
-0.25 dium at physiological pH and, hence, it can be used in
-0.3
live cell imaging studies with detection limit in the ppm
region for Cu2+ion in case of poisoning. The resultant
-0.35
-4.61 -4.56 -4.51 -4.46 -4.41 -4.36 -4.31 -4.26 Cu(II) complex is further being explored as a probe for
logCu2+ M-1 other substances and the investigation will be published
in due course.
 RSC Advances
ASSOCIATED CONTENT
Supporting Information
Experimental procedures, spectroscopic data and different
calculated data and graph (PDF). This material is available
free of charge on the RSC Publications website via the Inter-
net at http://pubs.rsc.org .
. Phys., 1945, 13, 507; (i) N. T. Southall, K. A. Dill, A. D. J.
8.
Published on 23 March 2016. Downloaded by University of California - Santa Barbara on 23/03/2016 11:23:24.
AUTHOR INFORMATION
Corresponding Author 9.
* Email: dhananjoym@yahoo.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors are greatly thankful to UGC for financial support
and Central University of Gujarat, Gandhinagar for instru-
mental and infrastructural facilities.
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