A dual functional colorimetric and fluorescence chemosensor based on benzo[f]fluorescein dye derivatives for copper ions and pH; kinetics and thermodynamic study

1. Accepted Manuscript
Title: A dual functional colorimetric and fluorescence
chemosensor based on benzo[f]fluorescein dye derivatives for
copper ions and pH; kinetics and thermodynamic study
Authors: Taha M. Elmorsi, Tarek S. Aysha, Oldˇrich
Machalick´y, Mahmoud B. Sheier, Ahmed H. Bedair
PII: S0925-4005(17)31104-8
DOI: http://dx.doi.org/doi:10.1016/j.snb.2017.06.084
Reference: SNB 22555
To appear in: Sensors and Actuators B
Received date: 5-12-2016
Revised date: 11-6-2017
Accepted date: 13-6-2017
Please cite this article as: Taha M.Elmorsi, Tarek S.Aysha, Oldˇrich Machalick´y,
Mahmoud B.Sheier, Ahmed H.Bedair, A dual functional colorimetric and
fluorescence chemosensor based on benzo[f]fluorescein dye derivatives for copper
ions and pH; kinetics and thermodynamic study, Sensors and Actuators B:
Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.084
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2. A highly sensitive colorimetric and fluorescence chemosensor based on xanthene dye
derivatives for copper ions and pH, kinetics and thermodynamic study
A dual functional colorimetric and fluorescence chemosensor based on
benzo[f]fluorescein dye derivatives for copper ions and pH; kinetics and thermodynamic
study
Taha M. Elmorsi1
*, Tarek S. Aysha2
, Oldřich Machalický 3
, Mahmoud B. Sheier1
and
Ahmed H. Bedair1
1
Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo,
Egypt, P.O.11651
2
Textile Research Division, National Research Centre, 33 El Bohouth st, Dokki, Giza,
Egypt, P.O.12622
3
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology,
University of Pardubice, Studentská 573, CZ-53210 Pardubice, Czech Republic.
*
Corresponding author: Taha M. Elmorsi (Assistance Prof. of Physical Chemistry)
E-mail : taha_elmorsi@azhar.edu.eg
Tel. 00201100045894
Address: Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City,
Cairo, Egypt, P.O.11651

3. Graphical Abstract

5. Highlights
 New derivatives of Xanthene dyes (BFFNH) was prepared and fully
characterized
 The prepared compound showing high selectivity and sensitivity in nano-mole
to Cu+2
 The BFFNH considered as a duel functional chemosensor to heavy metal and
pH
 Thermodynamics and kinetics study of the coordinated sensor was
investigated
 The application of BFFNH for monitoring of Cu2+
ions in water was studied

6. Abstract
A new dual functional fluorescein-based colorimetric chemosensor 3',6'-dihydroxy-2-
(((2-hydroxynaphthalen-1-yl)methylene)amino)-6-methyl-4-(p-
tolyl)spiro[benzo[f]isoindole-1,9'-xanthen]-3(2H)-one (BFFNH) derived from
benzo[f]fluorescein was synthesized. Spectroscopy analysis confirmed the structures
of the prepared compounds. BFFNH shows the colorimetric selectivity and sensitivity
towards the aqueous solution of Cu2+
ions with a detection limit in the micromolar
range (0.5 µM). The applications of BFFNH was extended for the detection of Cu2+
ions in real water samples (tab and synthetic water) with a high recovery %. Also,
BFFNH appeared to be highly sensitive with a quick response as fluorescence probe
to alkaline pH hence its pKa value calculated as 7.91. Thermodynamic parameters
ΔS
, ΔH
and ΔG
investigated at four temperatures (15, 20, 25 and 30 °C). Kinetic
study showed a first-order reaction with respect to the ligand. Also, the association
constant (Ka) of BFFNH which binding with Cu2+
ions based on 1:1 stoichiometry
was calculated.
Keyword
Benzo[f]fluorescein dyes, fluorescence probe, Heavy metals, Colorimetric
chemosensor, Kinetic and thermodynamic study.

7. 1. Introduction
Nowadays, a huge number of research are concerning with innovate and design a new
sensitive and selective at ultra-low concentration of colorimetric chemosensors and
fluorescence probes for toxic species detection. This observable priority is expected
due to its valuable requirement in health care and environmental conservancy for the
human prosperity in 21st
century.[1-3] Metals represent an important component of
the Earth’s crust which generally exist in trace concentrations in environmental
samples, where in humic substances have a complexation affinity toward them.
Copper ion, represent a very important transition metal ion for the human body,
assumes different roles in physiological processes and is a key component of an
extensive variety of enzymes such as copper-zinc superoxide dismutase, cytochrome c
oxidase, ceruloplasmin, lysyl oxidase, tyrosinase, dopamine b-hydroxylase and
peptidylglycine a-amidating monooxygenase.[4,5] Variations in normal copper levels,
both systemic as well as on a tissue or cellular scale, are embroiled in an extensive
variety of diseases such as Menkes disease, Wilson's disease, Alzheimer's disease,
Parkinson's disease and transmissible spongiform encephalopathy.[6-11] Furthermore,
copper ion is a significant environmental pollutant throughout the world due to its
across the board use in industry, agriculture, household utensils and water pipes.
Thus, it is of increasing importance to improve fast, proper and effective methods for
the qualitative and quantitative monitoring of the heavy metals.
The most advantage of the chemosensors based on color changes is that, the easy of
monitoring of the ions or the pH by naked eyes which does not need any complicated
instrumentation in additional to its inexpensive and high sensitivity.[12]
Because pH plays an essential role in different systems, especially within cells such as
apoptosis and cell growth, signal transduction and autophagy.[13-26] Abnormal
intracellular pH values indicate abnormal cell events and are observed in some
diseases including cancer and Alzheimer’s disease. [27] Numerous techniques for
estimation of pH values have achieved highly successful including microelectrodes,
[28] acid-base indicator titration, [29] potentiometric titration and fluorescent
probes.[30,31] One of the advantages of the new materials for ionic species sensors is
to import multifunctionality of these materials, for example; a dual chemosensor of a
selective ion and pH became a great target. Dual-function fluorescent chemosensors

8. designed for detecting different metal ions are plentiful, for pH and metal ions using
different optical signals are relatively few.[32,33] A considerable number of relevant
works concerning with colorimetric chemosensor for monitoring Cu2+
have been
reported,[34-36] and it was observed that most of the reported Cu2+
selective
colorimetric sensors different weakness such as poor detection limit, long response
time and interference from other transition metal ions.[37-42] Thus, developing more
efficient colorimetric chemosensing molecules for the naked-eye detection of Cu2+
in
an aqueous solution as well as the creation of a dual functional chemosensors is still
in required. Studies related to this area are of great challenge and continue to be of
great interest.
This work aimed to prepare benzo[f]fluorescein derivative (BFFNH) as a new
colorimetric chemosensor for metal ions. The selectivity, sensitivity along with the
detection limit of Cu2+
ions in aqueous medium were studied. Also, the effect of pH
on the fluorescence intensity of BFFNH studied in broad range of pH values (3.5-
10.9). In addition, thermodynamic and kinetics of the complex formation was
determined.
2. Experimental
2.1. Materials and apparatus
7-Methyl-1-p-tolyl-2,3-naphthalic anhydride (1) was prepared as described in the
literature,[43] resorcinol ≥ 99%, methanesulphonic acid ≥ 99.5% and Tris–HCl were
purchased from Sigma-Aldrich (Germany). Solvents such as Methanol, methylene
chloride were analytical grade while absolute ethanol was spectroscopy grade.
Britton–Robinson (B–R) buffer was prepared as reported.[44] All materials used
without any further purification. TLC performed using Aluminium-backed silica gel
plates (Merck, DC Kieselgel 60 F254). 1
H and 13
C NMR spectra were measured on a
Bruker DMX-400 spectrometer operating at 400, 101 MHz. Thermo Scientific ISQ
LT Single Quadrupole GC-MS in electron impact (EI) mode used for detecting the
mass spectra of prepared compounds. UV/Vis absorption spectra were recorded by a
Perkin-Elmer lambda 25 UV/Vis spectrophotometer. Fluorescence spectra were
studied by JASCO FP8300 spectrofluorometer. FT-IR spectra were measured on an
Agilent Cary 630 spectrometer. The pH measurements were carried out on a 3520 pH
Meter (JENWAY, England). Melting points were measured on a Stuart melting point
SMP30.

9. 2.2. Synthesis
The preparation procedure of colorimetric chemosensor BFFNH is based on a multi-
step reaction as shown in Scheme 1, at first 6-methyl-4-(4-
methylphenyl)benzo[f]fluorescein (BFF) was prepared as a key intermediate, then
refluxed with hydrazine hydrate to obtain 6-methyl-4-(p-tolyl)benzo[f]fluorescein
Hydrazide (BFFH),[45] which used as a final intermediate for prepare BFFNH.
2.2.1. Synthesis of BFF
In 100 mL three-necked round flask, 7-methyl-1-p-tolyl-2,3-naphthalic anhydride (1)
(3.02g, 10.00 mmol), resorcinol (2.20g, 20.00 mmol) with (15 mL) methanesulphonic
acid were mixed, stirred under nitrogen and heated in oil bath at 50 o
C for 5h. Then
the temperature was increased to 85 o
C for 48h. The mixture was cooled and poured
into a 100 mL of ice-water. The precipitate was filtrated off and washed with 5% HCl
solution. Further purification of the precipitate performed by dissolving in 5% NaOH
then precipitated by 5% HCl. The precipitate was filtrated and dried. The product (red
powder) of the BBF was obtained (4.33 g, 89 % yield, melting point >300 °C).
IR (KBr, cm-1
), vmax = 3767, 3718, 3638, 1727, 1659, 1591, 1464, 1381, 1229, 1205,
1109, 851.
Mass spectrum of BFF (C32H22O5) showed a molecular ion peak at m/z = 487 (20 %,
M+
).
1
H NMR (DMSO-d6, 400 MHz, δ, ppm): 2.36 (s, 3H), 2.59 (s, 3H), 6.95 (d, J = 7.5
Hz, 2H), 7.21 (dd, J = 7.5, 1.4 Hz, 1H) 7.29 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 1.4 Hz,
1H), 7.52 (d, J = 7.5 Hz, 2H), 7.59 (dd, J = 7.5, 1.4 Hz, 1H), 7.68 (s, 1H), 7.75 (s,
1H), 8.00 (d, J = 1.4 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.41 (s, 1H) and 10.17 (s, 2H,
2OH).
13
C NMR (DMSO-d6, 101 MHz, δ, ppm), 21.43, 22.17, 81.25, 102.53, 111.42,
112.43, 122.24, 124.43, 125.74, 128.63, 128.74, 128.91, 129.94, 130.84, 132.61,
132.91, 133.77, 136.32, 136.98, 137.00, 147.52, 152.78, 158.52 and 164.97.
2.2.2. Synthesis of BFFH
In a 100 mL round flask, BFF (4.33 g, 8.90 mmol) was dissolved in 20 mL of
methanol, followed by the addition of hydrazine monohydrate (17.61 mL, 0.36 mol).
The mixture was refluxed for 12h until the fluorescence of the solution disappeared.
The solvent and the excess of hydrazine hydrate was distilled off using rota-

10. evaporator under vacuum. A 100 mL of 0.1 N HCl was added to the oily residual.
The yellow precipitate was filtrated off and washed with 5% HCl solution. The crude
product was recrystallized from ethanol/H2O to give BFFH (3.52 g, 79%) as a bright
yellow crystal with Rf of 0.43 (CH2Cl2 : methanol,10:1) and melting point >300 °C.
IR (KBr, cm-1
), vmax = 3496, 3261, 3187, 3048, 2920, 1684, 1613, 1505, 1446, 1174,
1110 and 845.
Mass spectrum of BFFH (C32H24N2O4) showed a molecular ion peak at m/z = 500
(81%, M+
).
1
H NMR (DMSO-d6, 400 MHz, δ, ppm): 2.31 (s, 3H), 2.44 (s, 3H), 4.27 (s, 2H, NH2),
6.43 (dd, J = 8.6, 2.4 Hz, 2H), 6.53 (d, J = 8.6 Hz, 2H), 6.59 (d, J = 2.4 Hz, 2H), 7.31-
7.36 (m, 6H), 7.45 (s, 1H), 7.82 (d, J = 8.9 Hz, 1H) and 9.80 (s, 2H, 2OH).
13
C NMR (DMSO-d6, 101 MHz, δ, ppm), 21.48, 21.99, 63.62, 102.67, 111.66,
112.43, 122.24, 124.43, 125.74, 128.63, 128.74, 128.91, 129.94, 130.84, 132.61,
132.91, 133.77, 136.32, 136.98, 137.00, 147.52, 152.78, 158.52 and 164.97.
2.2.3. Synthesis of BFFNH
In a 100 mL round flask, BFFH (0.56 g, 1.00 mmol) and 2-hydroynaphthaldehyde
(0.17 g, 1.00 mmol) were dissolved in 25mL methanol with 0.5 mL of glacial acetic
acid and the mixture was refluxed for 7h. The solid precipitate was filtered off,
washed with cold methanol, and dried to obtain BFFNH, which was further purified
by recrystallization using methanol/ dichloromethane. The greenish-brown powder
was obtained (0.57g, 87% yield) with Rf of 0.50 (CH2Cl2 : methanol 10:1) and
melting point >300 °C.
IR (KBr, cm-1
), vmax = 3227, 3048, 2920, 1692, 1617, 1502, 1443, 1175, 1108, 992,
744.
Mass spectrum of BFFNH (C43H30N2O5), showed a molecular ion peak at m/z = 654
(21%, M+
).
1
H NMR (DMSO-d6, 400 MHz, δ, ppm): 2.33 (s, 3H), 2.45 (s, 3H), 6.47 (d, J = 2.4
Hz, 2H), 6.49 (d, J = 2.4 Hz, 2H), 6.70–6.72 (m, 3H), 6.73 (s, 1H), 7.03 (d, J = 8.9
Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 7.2 Hz, 3H), 7.42 (s, 1H), 7.43 (d, J =
2.9 Hz, 1H), 7.64 (s, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.88 (d, J
= 8.8 Hz, 2H), 9.76 (s, N=C–H, 1H), 10.04 (s, 2H) and 11.56 (s, 1H).

11. 13
C NMR (DMSO-d6, 101 MHz, δ, ppm): 21.48, 21.97, 64.33, 102.74, 109.27,
111.04, 113.17, 118.99, 121.38, 122.85, 123.76, 123.99, 125.92, 128.20, 128.87,
129.29, 129.37, 130.76, 131.72, 132.59, 132.93, 133.64, 134.52, 137.00, 137.42,
138.75, 146.17, 149.17, 152.63, 158.21, 159.10, 163.14.
2.3. Preparation of solutions for spectral measurements
To study cation selectivity, two separate solutions (with a concentration of 1.0 mM)
of both the BFFNH as a ligand (in absolute ethanol) and the metal salt solution (in
distilled water) were prepared. In this study we used different metal salt solutions
such as Ni(NO3)2.6H2O, Co(NO3)2.6H2O, Cr(NO3)3.9H2O, Cd(NO3)2.4H2O,
Fe(NO3)3.9H2O, KNO3, Ca(NO3)2.4H2O, Ba(NO3)2, NaNO3, Sr(NO3)2,
Mg(NO3)2.6H2O, Pb(NO3)2, AgNO3, MnCl2, Hg(CH3COO)2, AlCl3, ZnCl2 and
Ce2(SO4)3.
The complex (BFFNH-Cu) was formed by mixing 0.2 mL of each solution with the
addition of 1.0 mL of Tris-HCl buffer (10 mM, pH 7.2) in a 10 mL measuring flask.
Then the volume was completed to 10 mL by using 1:1 ethanol/water. The final
concentration of both the sensor and the metal salts in the prepared solution was 20
μM.
2.3.1. Stoichiometry, thermodynamic and kinetics study
The stoichiometric ratio of the coordination between BFFNH and Cu2+
ions was
studied using Job’s plot (which known as a continuous variation method). A stock
solution of equimolar (0.1 mM) of both the BFFNH and Cu2+
ions were mixed at
different volumes (from 0.2 to 2 mL). The molar ratio of BFFNH was varied from 0.1
to 0.9, while the total concentration of the BFFNH and Cu2+
ions remain constant in
each solution. The absorbance of BFFNH-Cu complex at 455 nm recorded and
plotted versus the molar ratio of BFFNH. In addition, the solutions of BFFNH-Cu
complex prepared for selectivity experiments were used to perform the required
experiments to calculate the association constant. Kinetic and thermodynamic studies
at different temperatures (15, 20, 25 and 30 C) were performed using 20 μM
solutions of BFFNH and Cu2+
ions to form the complex of BFFNH-Cu. The fast
change in the absorbance with a time of both BFFNH and BFFNH-Cu complex were
measured using the stopped-flow technique. All kinetic experiments were minimally
ten times repeated and averaged. Absorbance obtained in each kinetic run were
converted to concentrations using the following Equations (1) and (2):

12. Where; [BFFNH]t, [BFFNH]0 and [BFFNH-Cu]t are the actual and initial
concentrations of the BFFNH and the actual concentration of the BFFNH-Cu
complex respectively. Abs, Abs0 and Abs∞ are the actual, the starting and the final
absorbances of both BFFNH and BFFNH-Cu complex. Both the rate of formation of
BFFNH-Cu complex and the thermodynamic parameters ΔS
, ΔH
and ΔG
were
calculated.
2.3.2. Detection limit calculation
At the maximum absorbance (λmax) 455nm, the changing in the absorbance of
BFFNH-Cu complex (as prepared above) due to the changing in the concentration of
Cu2+
ions were recorded by UV-Vis spectrophotometer and the calibration curve was
plotted. The limit of detection (LoD) of Cu2+
ions using the prepared colorimetric
chemosensor and the limit of quantitation (LoQ) were calculated based on the
calibration curve method.[46]
2.4. pH fluorescence probe solutions
A series of different buffer solutions (pH from 3.5 to 10.9) in 1:1 ethanol: water were
prepared using Britton and Robinson universal buffer. A stock solution of 1.0 mM of
the BFFNH was prepared in absolute ethanol. The pH fluorescence probe solutions
(20 µM) were prepared by the addition of 0.2 mL of BFFNH in a 10 mL volumetric
flask and completed with the buffer solutions. The change in emission spectra was
detected using a fluorimeter.
3. Results and discussion
3.1. Synthesis of BFFNH compound
A multi-step reaction used for the synthesis of the BFFNH is described in a Scheme
1. In the first step, 6-methyl-4-(p-tolyl)benzo[f]fluorescein (BFF) produced by the
reaction of resorcinol with a 7-methyl-1-p-tolyl-2,3-naphthalic anhydride (1) in the
presence of catalytic amount of methansulphonic acid. As-prepared BFF formed in
three different isomers as shown in Figure 1. Hence the TLC monitoring of the BFF

13. show three different spots as a dull yellow (lactone isomer), yellow (zwitter ion
isomer) and red (p-quinonoid isomer) with very close Rf values [45,47,48]. Then the
BFF isomers (without isolation) were refluxed with hydrazine hydrate to obtain 6-
methyl-4-(p-tolyl)benzo[f]fluorescein hydrazide (BFFH) which reacts via a
condensation reaction with 2-hydroynaphthaldehyde to produce the probe BFFNH.
Scheme 1. The routes of synthesis of the probe BFFNH
Figure 1
3.2. Binding studies of BFFNH with Cu2+
(Reaction mechanism)
The mechanism of the coordination between BFFNH and Cu2+
ions confirmed by
UV/Vis and 1
H NMR spectroscopy.
3.2.1. UV/Vis spectral of BFFNH-Cu complex
UV/Vis spectra of BFFNH and BFFNH-Cu is shown in Figure 2. The addition of a
solution of Cu2+
ions to BFFNH solution leads to changing the color from colorless to
strong yellow. The coordination of BFFNH with Cu2+
ions led to both the extent of
the conjugation and the red shift of absorption spectra from 380 to 455nm. This
changing in the color is due to the ring opening of spirolactam ring in xanthene
moiety as shown in Figure 3(a).
Figure 2

14. 3.2.2.1
H NMR spectral of BFFNH-Cu complex
The 1
HNMR spectra of both BFFNH and BFFNH-Cu were determined in DMSO-d6.
These results were used to further confirm the mechanism of the coordination
between BFFNH and Cu2+
ions and the position of their bonds. Spectra of BFFNH
Figure 3(b) showed a clear band at 9.79, 10.04 and 11.56 ppm corresponding to
azomethane (–N=CH-), 2OH in xanthene moiety and OH group in naphthyl moiety
respectively. The addition of Cu2+
ions to the BFFNH with 1:1 molar ratio leads to a
significant change in the previously mentioned bands as shown in Figure 3(b). The
results showed a broadening in all proton signals which further confirms the
coordination reaction between BFFNH and Cu2+
ions. Furthermore, upon the addition
of Cu2+
ions also there was a significant downfield shift in the imino proton signal
(Ha) and the OH proton signals (Hc) of naphthyl moiety of BFFNH-Cu Figure 3(b).
This finding indicated that the coordination bonds formed due to the binding between
Cu2+
ions and both imino group (–N=CH-) and OH group of the naphthyl moiety.
Similar results were previously reported for the brooding and the downfield shift in
proton signals of different ligands coordinated with Cu2+
ions due to the paramagnetic
of the Cu2+
ions.
Figure 3
3.2.3. ESI-MS spectra of BFFNH-Cu complex
ESI-MS spectra of the reaction between BFFNH and Cu2+
is shown in Figure 4, (a)
the peak at m/z = 653 (M-H); 689 (M-H+2OH) for BFFNH only, (b) the peak at m/z =
653 (M-H); 689 (M-H+2OH); 716 (M-2H+Cu); 750 (M-H+2OH+Cu) for BFFNH +
0.5 equiv. Cu2+
and finally (c) the peak at m/z = 716 (M-2H+Cu); 750 (M-
H+2OH+Cu) for BFFNH (20 μM) with 1.0 equiv. of Cu2+
. The results indicated that
the stoichiometry of the BFFNH and Cu2+
is 1:1, which confirmed the value
calculated from Job’s plot (Figure 9).
Figure 4
3.2.4. IR Spectroscopy of BFFNH-Cu Complex
Figure S9 (supplementary data) showed IR spectra of BFFNH and BFFNH-Cu, a
clear characteristic absorption bands of BFFNH at 1692 cm-1
due to (˃C=O) lactam
in benzo[f]fluorescein moiety. Upon the formation of BFFNH-Cu complex, the

15. characteristic band showed a shift to lower frequency at 1611 cm-1
. This may be due
to executing the oxygen of the carbonyl group in coordination.[49] The band related
to azomethane (–N=CH–) appeared at 1617 cm-1
which showed a shift to lower
frequency in the case of BFFNH-Cu at 1521 cm-1
due to complexation including the
involvement of nitrogen of the azomethine group in coordination.[50] The band at
1175 cm-1
corresponding to (C–O) in naphthaldehyde moiety, which shifted at lower
frequencies in case of BFFNH-Cu at 1015 cm-1
due to coordination of the phenolic
oxygen of the BFFNH with Cu2+
ions.[51] Thus, it can be concluded that the BFFNH
is coordinating via the carbonyl group of lactam ring in benzo[f]fluorescein moiety,
the nitrogen of the azomethine group and the hydroxyl group of the naphthaldehyde
moiety.
3.3. The selectivity of BFFNH towards different metal ions.
The selectivity of the chemosensor was studied by preparing a 20 µM solution of
BFFNH in ethanol/water 1:1 in the presence of Tris-HCl buffer (10 mM, pH=7.2).
The pure BFFNH probe shows a maximum absorption (λmax) at 380 nm with
absorption coefficient () of 21500 1/(M.cm). When the BFFNH was mixed with 20
µM of different heavy metal solutions, a significant new absorption band appeared at
455 nm with absorption coefficient () of 20800 1/(M.cm) upon the addition of one
equivalent of Cu2+
ion solution only. While a minor interference bands appeared in the
case of addition of Co2+
and Ni2+
ion solutions only as shown in Figure 5.
Furthermore, the relative percent of absorbance of BFFNH complexes with different
metal ions to the absorbance of BFFNH-Cu complex is shown in Figure 6. We
noticed that the interference of Co2+
and Ni2+
ions was only 17 and 29% respectively.
This results confirmed the strong selectivity of BFFNH probe towards the Cu2+
ion
solution.
Figure 5
Figure 6
3.3.1 Effect of anion on BFFNH
The selectivity of the BFFNH towards copper anions was also studied by using
different copper anion such as nitrate, sulphate, acetate and chloride. BFFNH has the
same maximum absorption λmax= 455 nm in UV/Vis spectra with CuSO4, Cu(NO3)2,

16. Cu(CH3COO)2, and CuCl2. So, no effect of anions on BFFNH as showed in Figure
S10 (supplementary data) was recorded.
3.3.2 Tolerance of BFFNH to Cu2+
over other metal ions
The sensitivity of BFFNH towards Cu2+
in presence of different metal ions in the
solution was studied as shown in Figure 7. The concentration of BFFNH was kept at
(20 μM) in Tris– HCl (10 mM, pH = 7.20) as aqueous buffer solution and the
absorption spectra was measured in case of the presence of different metal ions such
as Na+
, K+
, Mg2+
, Ca2+
, Ba2+
, Mn2+
, Al3+
, Cr3+
, Co2+
, Ni2+
, Ag+
, Zn2+
, Hg2+
, Sr2+
, Ce3+
,
Cd2+
, Fe3+
and Pb2+
. The results indicated that monitoring of Cu2+
by BFFNH was not
affected by other coexisting metal ions.
Figure 7
3.3.3 The effect of pH change on monitoring of copper ions by BFFNH
colorimetrically
The effect of pH on BFFNH in the absent and presence of Cu2+
at 455nm was showed
in Figure 8, colorimetric study of the pH effect showed no significant change in
absorption spectra at 455nm in the absence of copper ions. While in the presence of
Cu2+
the increase in the pH value above 7, the hypochromic effect become significant.
This results may ascribed to the formation of Cu(OH)2 which interrupting the
formation of copper complex with BFFNH. Thus, BFFNH can be used for the
detection of Cu2+
in aqueous medium under neutral conditions.
Figure 8
3.4. Determination of stoichiometry and Association constant
3.4.1. Determination of stoichiometry using Job’s plot method
The molar ratio of the coordination between BFFNH was studied using the Job’s plot
method[52] as shown in Figure 9. In this method, each experiment performed with
different concentrations of both BFFNH and Cu2+
ions with maintaining the total
concentration at 20 µM. The plot obtained by measuring the absorbance at 455 nm for
nine experiments with molar fraction of BFFNH from 0.1 to 0.9. The maximum
absorbance intensity at 455 nm appeared when the molar fraction of BFFNH was 0.5,
which indicated that the 1:1 stoichiometry was the possible binding mode of BFFNH
with Cu2+
ions.

17. Figure 9
3.4.2. Calculation of the binding association constant
The association constant was calculated using the Benesi-Hildebrand plot (or a
double-reciprocal plot) as shown in Equation (3):[53]
where; A is the experimentally measured absorption intensity, A0 is the absorption
intensity of free BFFNH, and Amax is the saturated absorption intensity of the
BFFNH-Cu complex. The association constant (Ka) was graphically evaluated by
plotting 1/[A – A0] versus 1/[Cu2+
], as shown in Figure 10. From the obtained linear
relationship, the binding constant was calculated which equal 3.16 × 104
M-1
.
Figure 10
3.5. Kinetic and thermodynamic study
BFFNH-Cu was formed using 1:1 molar ratio of BFFNH and Cu2+
ions at a different
temperature to determine its rate and the thermodynamic parameters. The change in
the concentrations of both BFFNH and BFFNH-Cu chemosensor with the changing
in the time recorded in the period of 20 seconds Figure 11(a). The results show that
the rate of decay of the reactant (BFFNH) is very similar to the rate of growth of the
product (BFFNH-Cu) indicating the first-order process with respect to BFFNH. This
finding is further confirmed by applying the two Equations (4) and (5) of first-order
and second-order models which were evaluated based on the value of the coefficient
of determination (R2
).
The results of Figure 11(b) and (c) indicated that the rate of the reaction was
favourable with the first-order model with respect to BFFNH as indicated by the high
value of (R2
) Table (1).

18. Table (1)
The rate constant of the reaction Table (2) enhanced slightly by increasing the
temperature from 15 to 30 C Figure 12(a). The relation between the rate constant
and temperature was plotted according to Arrhenius Equation (6) as shown in Figure
12(b).
where; k is the rate constant (s-1
) Ea is the activation energy (kJ/mol), R is the
universal gas constant (0.008314 kJ/mol. K), T is the absolute temperature (K) and A
(s-1
) is the pre-exponential factor (which it is the number of collisions per second
occurring with the proper orientation.
Table (2)
The values of both Ea and A were obtained from the slope and the intercept of Figure
11(c) respectively and tabulated in Table (2). The obtained value of Ea is very low
(2x10-2
kJ/mol) which indicated the fast transfer of reactants to products with minimal
energy. These results confirmed the fast response of the chemosensor towards Cu2+
ions at room temperature.
Thermodynamic parameters including the change in the standard enthalpy (ΔH
), the
change in the standard entropy (ΔS
) and the change in the standard free energy (ΔG
)
determined for the formation of BFFNH-Cu chemosensor at 15, 20, 25 and 30 °C.
The values of (ΔG
) were calculated from Van't Hoff isochore Equation (7). The
values of and calculated from the slope and the intercept respectively of
Figure 12 according to Equation (7). The negative values of Table (3) indicated
that the formation of BFFNH-Cu chemosensor is spontaneous. Also, the positive
values of both (41.34 kJ/mol) and (0.26 kJ/mol. K) are an indication of the
endothermic nature of the formation process along with a minimal increase in the
randomness of the lattice of BFFNH-Cu chemosensor.

19. Table (3)
Figure 11
Figure 12
3.6. Determining the sensitivity and the limit of detection
The data of the calibration curve, which performed to calculate the concentration of
the formed BFFNH-Cu complex, was used as an indication of the sensitivity of
BFFNH to copper ions. In this procedure, different concentrations of Cu2+
ions (0.0-
2.0 equivalent) were added to 20 μM of BFFNH using Tris-HCl buffer. The
absorption spectra showed an increase in the absorption band at 455 nm by increasing
the amount of Cu2+
ions and the spectrum passed through two isosbestic points at 352
and 410 nm (Figure 13). This increase in the absorbance and two isosbestic points is
confirming the sensitivity of BFFNH to Cu2+
ions in a solution and the formation of a
new BFFNH-Cu complex in equilibrium with the free BFFNH.[54] On the other
hand, the limit of detection (LoD) and limit of quantitation (LoQ) were calculated in this
study by the method of the calibration curve. It is known that the lowest amount of an
analyte in a sample which may detected without enough precision and accuracy called the
LoD. While the lowest amount which can be detected with enough accuracy and precision
is called LoQ. The absorbance values at 455 nm were plotted versus the Cu2+
ion
concentrations (Figure 14) to obtain the slope and the intercept of the calibration
curve. Then the slope and the intercept were used to calculate LoD and LoQ as in
Equations (8) and (9).[55]
where, is the residual standard deviation (error) of a regression line and S is the
slope (16208 M-1
) of the calibration curve. The value of was calculated as
0.002717. The value of the limit of detection and limit of quantitation of Cu2+
ions by
BFFNH were calculated as 0.50 µM and 1.68 µM respectively. The results of the
value of LoD of BFFNH chemosensor for Cu2+
ion was compared with the literature

20. (based on spiro-xanthene derivatives) as shown in Table (4). It can be noted that
BFFNH has a very high sensitivity towards Cu2+
ions in the range of micro-molar
concentration in comparing to other derivatives.
Figure 13
Figure 14
3.7. Recovery and Reversibility with EDTA
The chelation of Cu2+
ions with BFFNH lead to the appearance of a yellow color
which resulted due to the opening of the spirolactam ring. The absorbance of the
formed complex (BFFNH-Cu) at 455 nm reached a maximum at 1:1 molar ratio of
BFFNH: Cu2+
ions. Upon addition of 4 equiv. of EDTA, which results in re-
disappearance of the color band at 455 nm. The reversible cycle could be repeated
several times by alternating the addition of Cu2+
ions and EDTA solution. Thus, the
changes in the absorbance intensity of BFFNH in Tris-HCl buffer solution (10 mM,
pH= 7.2) are controlled by the two spices, Cu2+
ions and EDTA solution. Hence Cu2+
ions caused yellow color, while EDTA returned the solution to colourless. The yellow
color disappeared and the absorbance at 455nm decreased dramatically to the original
absorbance of free BFFNH indicating that the coordination of BFFNH with Cu2+
is
chemically reversible[54] as shown in Figure 15.
Figure 15
The reversibility and reproducibility was observed up to 5 cycles as shown in Figure
16.
Figure 16
3.8. Fluorescence spectroscopic studies of BFFNH as a pH Probe
The emission spectra of BFFNH in absence and presence of OH-
ions in alkaline
medium (B-R buffer at pH = 10.9) and with the presence of various metal ions at pH
= 7.2 is shown in Figure 17(a). The results indicated that both the solution of
BFFNH alone and/or in the presence of different metal ions did not produce any
significant changes in the emission intensity. While in the case of increasing the
solution pH up to 10.9, the emission spectrum was significantly increased. The
presence of high concentration of OH-
ions in the alkaline medium would lead to the

21. deprotonation of BFFNH and open the spirolactam ring (imido anion) as shown in
Figure 17(b). The deprotonation of BFFNH resulted in the presence of the negative
charge onto the xanthene moiety which increases the conjugation and caused a high
increase in the fluorescein intensity. Furthermore, the sensitivity of BFFNH probe for
pH was investigated by the fluorescence titration of BFFNH with different pH values
(3.5-10.9) using B-R buffer (Figure 19(a)). The data showing a significant increase of
the emission intensity by increasing the pH value till 10.9 and no significant was
observed over this value.
Figure 17
3.8.1. Effect of time in the detection of pH using BFFNH probe
The effect of time on fluorescence intensity of BFFNH (20µM) when the pH
increased to 10.9 is shown in Figure 18. After 30 seconds, the fluorescence intensity
reached its maximum value then remain constant, that means BFFNH probe shows a
very high sensitivity towards alkaline medium as fluorescence probe.
Figure 18
3.8.2. Fluorescence titration and acidity constant calculation (pKa)
Furthermore, the sensitivity of BFFNH probe for pH was investigated by performing
a fluorescence titration for BFFNH at different pH values (3.5-10.9). The
fluorescence intensity (Figure 19(a)) shows a minimal change in acidic medium at
pH values from 3.5 to 6.9. However, increasing the pH values from 7.9 to 10.9 led to
a high increase in the fluorescence intensity. Photography of representative samples
for the change in both the color and the fluorescence intensity of BFFNH at different
pH values are shown in the inset of Figure 19(a). The fluorescence intensity of
BFFNH with different pHs at 520nm used to calculate the acidity constant pKa value
of BFFNH, using Henderson-Hasselbalch Equation (10):[56]
where; I is the observed fluorescence intensity at a fixed wavelength, Imax and Imin are
the corresponding maximum and minimum intensity respectively.
The sigmoidal curve (Figure 19(a)) shows a very weak emission spectra of BFFNH
at a pH value less than 6.90. The low concentration of OH-
ions in that range of pH, is

22. Accepted Manuscript
Title: A dual functional colorimetric and fluorescence
chemosensor based on benzo[f]fluorescein dye derivatives for
copper ions and pH; kinetics and thermodynamic study
Authors: Taha M. Elmorsi, Tarek S. Aysha, Oldˇrich
Machalick´y, Mahmoud B. Sheier, Ahmed H. Bedair
PII: S0925-4005(17)31104-8
DOI: http://dx.doi.org/doi:10.1016/j.snb.2017.06.084
Reference: SNB 22555
To appear in: Sensors and Actuators B
Received date: 5-12-2016
Revised date: 11-6-2017
Accepted date: 13-6-2017
Please cite this article as: Taha M.Elmorsi, Tarek S.Aysha, Oldˇrich Machalick´y,
Mahmoud B.Sheier, Ahmed H.Bedair, A dual functional colorimetric and
fluorescence chemosensor based on benzo[f]fluorescein dye derivatives for copper
ions and pH; kinetics and thermodynamic study, Sensors and Actuators B:
Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.084
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
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apply to the journal pertain.

23. the low values of RSD. This finding offered the possibility of application of BFFNH
as a sensor for detecting Cu2+
ions in real water samples.
4. Conclusion
In summary, the BFFNH was designed and synthesised as dual-function fluorescent
chemosensor derivative from benzo[f]fluorescein. BFFNH shows a very high
selectivity and sensitivity to Cu2+
ions in the micromolar range (0.5 µM). Copper ions
binded with BFFNH in 1:1 molar ratio. Results indicated that the BFFNH can be
used for visualisation detection of Cu2+
in Tab and wastewater samples. The high
value of Ka (3.16×104
M-1
) indicated the formation of a high stable complex of
BFFNH-Cu which reached equilibrium in less than 15 seconds. The kinetic and
thermodynamic study showed that the BFFNH-Cu formed spontaneously with a high
rate constant (k1 = 0.18 s-1
). The change in the temperature has a minimal effect on the
formation of BFFNH-Cu complex. On the other hand, BFFNH also exhibited a very
significant sensitivity and selectivity along with a quick response to alkaline pH as
indicated by its pKa value of 7.91.
Acknowledgment
We thanks the Chemistry Department, Faculty of Science, Al-Azhar University. We
thanks also National Research Centre, Giza, Egypt through project 11070103
(colorimetric chemosensors and fluorescence probes: a promising strategy for
environmental monitoring of ionic species and toxic gases) for the financial support.

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30. Dr. Taha M. Elmorsi, Ph.D graduated from Faculty of science, Al-Azhar University,
Nasr city, Cairo, Egypt with a B.Sc degree in chemistry in 1989. He finished his
master Degree at Al-Azhar University, Chemistry department, Nasr city, Cairo, Egypt
in 1993. He got his ph.D from Physical and Environmental Chemistry department,
University of Manitoba, Winnipeg, MB, Canada in 2003. He joined his post-doctoral
position in the area of photocatalysis “A Second Generation Photocatalytic Oxidation
Processes for the NASA Advanced Water Recovery System” in 2007.
In 2009 he cooperate with University of Aberdeen, Scotland, UK. In the area of
Nanotechnology for Sustainable Water Purification.
He is currently work as Associate professor, Chemistry Department, Faculty of
Science, Al-Azhar University, Nasr city Cairo, Egypt.
Dr. Tarek Aysha, Ph.D was born on 1980 in Elgharbia, Egypt. He graduated from
Faculty of Science, Benha University, Egypt in 2001, with a B.Sc. degree in
chemistry. He finished his M.Sc. degree from Faculty of Science, Benha University,
Egypt in 2007 in organic chemistry. He started his scholarship for studying Ph.D. at
Faculty of Chemical Technology, University of Pardubice, Czech Republic in 2008
and he got his Ph.D. degree in 2011 in the field of organic chemistry and technology.
He started his post-doctoral at university of Pardubice, Czech Republic from 1-07-
2012 for three years. He is currently working as a researcher at Textile Research
Division, Department of Dyeing, Printing &Textile Auxiliaries, National Research
Centre, Dokki, Giza, Egypt since 2003. He a warded the Prize of the dean of Faculty
of Chemical Technology, University of Pardubice, Czech Republic for the excellent
Ph.D. thesis for academic year 2010/2011. He awarded the prize of the pioneer from
national research centre, Dokki, Giza, Egypt for the year 2015. His research interests
are concerning with functional dyes, colorimetric chemosensor and fluorescence
probe, dye sensitizer solar cell and high performance colorants for textile and non-
textile applications.
Dr. Oldrich Machalicky was born on 1959 in Jaromer, Czech Republic. He
graduated from Faculty of Chemical Technology, University of Pardubice, Czech
Republic in 1992. Doctoral thesis he defended in 1998 at the Department of

31. Technology of Organic Substances, University of Pardubice and he worked there as
an assistant professor from 1999 to 2008. Now he works as assistant professor at the
Institute of Organic Chemistry and Technology of University of Pardubice. The field
of his scientific interest is organic synthesis, heterogeneous catalysis and
photochemistry.
Dr. Mahmoud B. Sheier, M.Sc; was born in 1987 in Beni Suef, Egypt. He graduated
from Faculty of science, Al-Azhar University, Nasr city, Cairo, Egypt with a B.Sc
degree in chemistry in 2008. He finished his master Degree at Al-Azhar University,
Chemistry department, Nasr city, Cairo, Egypt in 2014 in “synthesis and evaluation of
some acid dyes and its utilization in textile coloration”. He studies now has PhD. at
Faculty of science, Al-Azhar University, Nasr city, Cairo, Egypt in the area of
fluorescence and colorimetric chemosensors. He currently works as an assistant
lecturer at Faculty of science, chemistry department, Al-Azhar University, Nasr city,
Cairo, Egypt.
Dr. Ahmed H. Bedair was born in 1948 in Monufia, Egypt. He graduated from Faculty of
science, Ain Shams University, Egypt with a B.Sc degree in special chemistry in
1970. He finished his master Degree at Al-Azhar University, Chemistry department,
Nasr city, Cairo, Egypt in 1975. He got his ph.D from Al-Azhar University,
Chemistry department, Nasr city, Cairo, Egypt in 1977.
He is currently work as Full professor, Chemistry Dept, Faculty of Science, Al-Azhar
University, Nasr city Cairo, Egypt.

32. Figure 1. The suggested isomers of BFF compound.

33. Figure 2. UV/Vis spectra of BFFNH (20 µM) in absence and presence of Cu2+
ions
(20 µM) in Tris-HCl buffer (1 mM, pH= 7.2).

34. Figure 3. (a) The changing in the color due to the ring opening of spirolactam ring.
(b) 1
H NMR spectra of BFFNH and BFFNH-Cu in DMSO-d6.

35. Figure 4. ESI-MS spectra of reaction between BFFNH and Cu2+
: (a) BFFNH (20
μM), (b) BFFNH (20 μM) with 0.5 equiv. of Cu2+
, (c) BFFNH (20 μM) with 1.0
equiv. of Cu2+
.

36. Figure 5. UV/Vis absorption spectra of BFFNH (20 µM) with different metal ions
(1:1 molar ratio) in Tris-HCl buffer (1 mM, pH= 7.2).

37. Figure 6. Relative percent of absorbance of BFFNH complexes with different metal
ions to the absorbance of BFFNH-Cu complex in Tris-HCl buffer (1 mM, pH = 7.2),
[Mn+
] = 20 µM.

38. Figure 7. Interference of binary solution of various metal ions toward BFFNH at
λmax= 455 nm in Tris– HCl (10 mM, pH = 7.20).

39. Figure 8. Absorption at 455nm of free BFFNH (20µM) and BFFNH-Cu (20µM) in
Tris–HCl buffer solution with different pH conditions.

40. Figure 9. Job’s plot of equimolar solution of BFFNH-Cu complex in 1:1
ethanol/water at 455 nm, ([BFFNH]+[Cu2+
]) = 20 µM).

41. Figure 10. Benesi-Hildebrand plot of 1/(A-A0) versus 1/[Cu2+
].

42. Figure 11. (a) Change in concentrations of BFFNH and BFFNH-Cu with time, (b)
First-order model, (c) Second-order model.

43. Figure 12. (a) Effect of temperature on the rate of the reaction, (b) plot of Arrhenius
equation and (c) plot of Van't Hoff isochore equation.

44. Figure 13. UV/Vis absorbance titration of BFFNH (20 µM) with different
concentration of Cu2+
ions (0-40 µM).

45. Figure 14. UV/Vis absorbance spectra at 455nm of BFFNH (20µM) with different
concentration of Cu2+
in Tris-HCl buffer (0.01M, pH=7.2).

46. Figure 15. (a) Absorbance spectra showing reversibility of BFFNH with Cu2+
by the
addition of various equivalents of EDTA and (b) suggested mechanism of binding of
BFFNH with Cu2+
ions.

47. Figure 16. The reversible and reproducible absorbance controlled by alternate
addition of Cu2+
ions and EDTA in Tris-HCl buffer solution (10 mM, pH= 7.2) of
BFFNH (20 μM).

48. Figure 17. (a) Fluorescence spectra of BFFNH with the presence of OH-
ions at
pH= 10.9 and with the presence of various metal ions at pH=7.2 and (b) Suggested
mechanism for spirolactam ring opening by OH-
ions.

49. Figure 18. The time of fluorescence intensity of BFFNH at λem= 480 nm (20µM) in
B–R buffer solution (pH = 10.9).

50. Figure 19. (a) Fluorescence spectra of BFFNH with different pH values, the inset
shows represented photography of BFFNH at different pH (1) Changes in color. (2)
Changes of Fluorescent intensity and (b) Fluorescence titration at 520 nm in different
pHs, the inset shows the linear relationship of fluorescence intensity from pH 6.9-8.9.
(B-R buffer, λex= 480 nm, pH (3.5-10.9).

51. Table (1). Parameters of linear form of first-order and second-order models
Temp.
(o
K)
linear first-order linear second-order
[BFFNH]0
(M)
Adj.
R2
t1/2
(s)
k1
(s-1
)
[BFFNH]0
(M)
Adj.
R2
t1/2
(s)
k2
(M-1
s-1
)
293 2.153 ×10-5
0.988 3.67 0.18 3.119×10-6
0.750 5.64 56840
Table (2). Parameters of the Arrhenius Equation
Temp.
(K0
)
k
(sec-1
)
Adj. R2
Ea
kJ/mol
A
(sec-1
)
283 0.168 0.990
0.002 9.0255 × 10+17288 0.176 0.989
293 0.184 0.988
298 0.209 0.960
303 0.232 0.957
Table (3). Thermodynamic parameters of BFFNH-Cu chemosensor formation
Temp.
(K0
) kJ/mol kJ/mol. K kJ/mol
288 14.39 -34.45
0.26 41.34
293 14.46 -35.22
298 15.05 -37.28
303 15.14 -38.14

52. Table (4). Comparison results with similar compounds based on spiro-xanthene derivatives as chemosensors for Cu2+
ion
Structure of
chemosensor
Media
Time
responsory
Detection limit Reproducibility Reference
HEPES (0.1 M, pH = 7.2)
in H2O/CH3CN (3:2, v/v) solution
5 min. 0.11 μM Reversible [57]
B–R buffer (pH = 9.0) 2 min. NA Reversible [58]
DMSO/H2O (4:6, v/v) 2 min. NA Reversible [59]
DMSO/HEPES (3:1, v/v, 1 mM, pH 7.2) solution 60 s. 0.296 μM NA [53]
Palitzsch’s buffer (0.1 M, pH 7.0) in 10% (v/v) THF
1 min. 0.45 μM Reversible [60]

53. CH3CN–H2O (3:7, v/v), pH 7.0 1 min. NA Reversible [61]
DMSO/Tris–HCl buffer (1:9, v/v, pH 7.0) solutions
< 30 s.
3.85 μM Reversible [62]
Tris–HCl buffer (10 mM, pH 7.0) in ethanol : water (1:1) solution
< 15 s.
0.5 μM Reversible This work
NA: not available.

54. Table (5). Determination of Cu2+
in tab and synthesised water samples at low and
high concentrations.
Sample
[Cu2+
] added
(µM)
[Cu2+
] found
(µM)
%
Recovery
%
RSDa
Tab water (blank) 0.0
Not
Detectable
---- ---
Tab water (with Cu2+
)
2 2.21 110 4.1
20 21.1 105 5.8
Synthesized waterb
2 2.19 99 2.7
20 20.8 94 4.7
a
ո=3.
b
Synthesized water prepared from tab water with 2 µM of Cu2+
+ 20 µM of (Ag+
+ Zn2+
+ Mn2+
+ Cd2+
+ Fe3+
+ Mg2+
) + 200 µM of (Na+
, K+
, Ca2+
+ Mg+
) in
presence 20 µM of BFFNH and 1 mM Tris-HCl buffer as pH=7.2.