Design of novel anthracene-based fluorescence sensor for sensitive and selective determination of iron in real samples

Article abstract of DOI:10.1016/j.jphotochem.2020.112819

A dipodal fluorescence (FL) system based on an anthracene platform was prepared by a general synthetic strategy. The water-miscible receptor was characterized by MALDI-MS, ¹H NMR, ¹³C NMR, and FT-IR. In order to the illustration of photophysical properties of the synthesized sensor, the excitation–emission matrix (EEM) analysis and 3D-fluorescence measurements were performed. Ultraviolet-Visible (UV–vis) and FL spectroscopies were carried out to evaluate the FL performance of the sensor. Low concentrations of iron ions (Fe³⁺) could be determined by this sensitive and simple sensor. Due to the quenching effect of Fe³⁺, a limit of detection (LOD) of 0.314 μM was obtained in the linear range of 0.3–130 μM. This fluorescent sensor was applied for measuring ferric ions in environmental water and tablet samples, which was highly efficient. The proposed strategy could be applied to develop sensors for not only for Fe³⁺ but also for many various metal ions using a diversity of fluorophores and ionophores.

Full text of DOI:10.1016/j.jphotochem.2020.112819

Journal Pre-proof
Design of novel anthracene-based fluorescence sensor for sensitive and
selective determination of iron in real samples
˘
Su¨reyya Oguz Tu¨may, Mahsa Haddad Irani-nezhad, Alireza
Khataee
PII:
S1010-6030(20)30617-1
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112819
JPC 112819
Reference:
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Journal of Photochemistry & Photobiology, A: Chemistry
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21 July 2020
28 July 2020
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Design of novel anthracene-based fluorescence sensor for sensitive
and selective determination of iron in real samples
Süreyya Oğuz Tümaya,1, Mahsa Haddad Irani-nezhadb,2, Alireza Khataeeb,c,3*
a Department of Chemistry, Gebze Technical University, 41400 Gebze, Turkey
b
Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of
Applied Chemistry, Faculty of Chemistry, University of Tabriz, 51666-16471 Tabriz, Iran
c Department of Environmental Engineering, Gebze Technical University, 41400 Gebze, Turkey
* Corresponding author:
a_khataee@tabrizu.ac.ir
Graphical abstract

Research highlights




Naphthalene appended dipodal anthracene sensor (1) was synthesized via a simple click
reaction.
Novel fluorescence sensor (1) demonstrated high sensitivity and selectivity towards Fe3+
over other competitive ions.
The spectrofluorimetric determination of iron in environmental water and tablet samples
was performed with using fluorescence sensor (1).
The developed spectrofluorimetric method showed a good aggrement with ICP-OES
analysis and spiked/recovery tests.
1

Abstract
A dipodal fluorescence (FL) system based on an anthracene platform was prepared by a general
synthetic strategy. The water-miscible receptor was characterized by MALDI-MS, 1H NMR, 13C
NMR, and FT-IR. In order to the illustration of photophysical properties of the synthesized sensor,
the excitation–emission matrix (EEM) analysis and 3D-fluorescence measurements were
performed. Ultraviolet-Visible (UV-Vis) and FL spectroscopies were carried out to evaluate the
FL performance of the sensor. Low concentrations of iron ions (Fe3+) could be determined by this
sensitive and simple sensor. Due to the quenching effect of Fe3+, a limit of detection (LOD) of
0.314 µM was obtained in the linear range of 0.3-130 µM. This fluorescent sensor was applied for
measuring ferric ions in environmental water and tablet samples, which was highly efficient. The
proposed strategy could be applied to develop sensors for not only for Fe3+ but also for many
various metal ions using a diversity of fluorophores and ionophores.
Keywords: Dipodal receptor; Anthracene; Fluorescent sensor; Environmental water; Tablet.
2

1. Introduction
Fundamental roles in humans, plants, and animals are done by dissolved ions in aqueous media.
Ions propagate the electronic signals and preserve the balance between intracellular and
extracellular environments fluids that is very important for various processes such as muscle
function, nerve impulses, regulation of pH level, and hydration [1]. The iron due to its important
role in electron transfer, DNA, RNA (synthesis and repair), O2 uptake, O2 metabolism, and enzyme
catalysis, is one of the most necessary paucity elements for life system [2]. Serious illnesses like
Parkinson’s and Alzheimer’s diseases and cancers are caused by iron accumulation, while iron
deficit leads breathing problems, diabetes, anemia, kidney, heart, and liver failure [3].
Although various instrumental methods have been applied in the iron detection including
atomic absorption spectroscopy [4], ion pair chromatography [5], electrothermal atomization
atomic absorption spectrometry (ETAAS) [6], voltammetry [7], and inductively coupled plasma
mass spectrometry (ICP-MS) [8], in environmental, biological, and industrial samples, but they
cannot be available in-field and achieved continuous monitoring. Also, they need pretreatment
processes and some reagents like eluents and adsorbents [9]. These disadvantages have limited
their applications. Therefore, the development of a determination method which solves the
problems still is a challenge for researchers. Due to its easy operation, high sensitivity, rapid
response, high efficiency, and remarkable selectivity, fluorescence (FL) is one of the most famous
and appropriate approaches for the detection of iron in aqueous or organic media [10].
Fluoroionophore system is a part of fluorescent sensor systems which consists of the fluorophore
and ionophore groups. The ionophore part interacts with metal ions while fluorophore moiety
converts the diagnosis occurrence received from ionophore part to analytical signals [11].
Synthetic receptors provide wide range of properties in proportion of the targeted metal ions’ shape
3

or size which is still a challenge for researchers to design new synthetic receptors with high
selectivity. In target component detection by interaction/complexation of ion with receptor,
synthetic receptor’s topology has an especial role [12].
Until now, manifold fluorescent sensors have been reported for Fe3+ detection based on both
fluorophore and ionophore systems however most of them because of low selectivity, high limit
of detection (LOD), or narrow linear range are not so suitable. Kang and Kim [13] synthesized a
chemosensor via the reaction of 2,3-dihydroxybenzaldehyde and octopamine for the detection of
iron. This water-soluble and bio-friendly sensor has been used for detection of Cd2+ and Zn2+ by
different FL emission (blue and green respectively). In another study, Gupta et al. [14] reported
2+
two fluorescence and colorimetric aminopyridine Schiff bases. They detected Fe3+, UO2 , Ni2+,
and Zn2+ via electrochemical, emission, and absorption properties of 2-((5-methylpyridin-2-
ylimino)methyl)phenol and 1-((5-methylpyridin-2-ylimino)methyl)naphthalen-2-ol. Kim et al.
[15] also developed a chemosensor for colorimetry detection of iron by changing the color from
colorless to dark green. This benzimidazole-based sensor could be recycled by treating with
ethylenediaminetetraacetic acid (EDTA).
Anthracene and its derivatives as fluorescent fluorophores, because of their easy process
ability, unique properties, and commercial availability, have been applied in various functions such
as sensing pH, small organic molecule, metal ions, and simple inorganic anions. Superior excimer
and monomer emission variations occur via importing donor/acceptor units into anthracene
backbones which is related to the space of two anthracene segments. So, tunable optical properties
could be attained by controlling the shape/size of the materials [16]. Due to its low FL quantum
yield [17] and short FL lifetime [18], naphthalene is of particular interest to be used as a donor or
an acceptor [19].
4

Discerning different colures of weak light is the trait of human eye which eliminates using an
expensive detector. These all results prompted us to prepare a novel fluorescent colorimetric sensor
by “Click” reaction. So that, 1, 2, 3 triazole serves as a bridge between fluorophores (naphthalene
and anthracene part) and acts as an ionophore to binds with analyte. The synthesized sensor (1)
provides different FL colures when reacted with iron which can be recognized by the naked-eye
upon UV light radiation. After the characterization of compound 1 via MALDI-MS, Nuclear
magnetic resonance spectroscopy (1H NMR and 13C NMR), and Fourier transform infrared
spectroscopy (FT-IR) methods, the excitation-emission matrix (EEM) analysis and 3D-FL
measurements were carried out to illustration of its photophysical properties. Finally, compound
1 has been applied for the determination of Fe3+ in environments, industrial, and biological
samples. Also, the validation of the sensor has been carried out via ICP-OES analysis and spike
and recovery tests.
2. Experimental
2.1. Reagents
Tetrahydrofuran (THF), 2-naphthol, sodium hydride (NaH), copper(I) bromide, deuterated
chloroform
(CDCl3),
dimethyl
sulfoxide
(DMSO-d6),
N,N,N’,N’’,N’’-
pentamethyldiethylenetriamine (PMDTA), and N,N- dimethylformamide (DMF) were purchased
from Sigma-Aldrich. Anthracene, n-hexane, paraformaldehyde, hexadecyltrimethylammonium
bromide, sodium azide, dichloromethane, ethanol, glacial acetic acid, hydrogen bromide,
acetonitrile (ACN), 2-bromoethanol were purchased from Merck and 2,5-dihydroxybenzoic acid
which was used as MALDI matrix was obtained from Fluka. Washing with n-hexane was applied
to purified NaH (>60%) because it is commercially available in mineral oil. Before using THF,
5

sodium/potassium alloy was used for distillation process. Inert argon or nitrogen atmosphere was
used for all synthetic processes. Silica gel (70–230 mesh, Kieselgel 60) and silica gel plates/F254
(0.25 mm thickness, Kieselgel 60) were used for chromatography processes and they were
purchased from Merck.
2.2. Equipments
The steady-state FL experiments and spectrofluorimetric analysis of real samples were
performed by Cary Eclipse spectrofluorometer (Varian, USA). Fluorolog 3-2iHR with Fluoro
Hub-B Single Photon Counting Controller (Horiba Jobin Yvon, France) was used for time
resolved-FL, 3D-FL emission experiments and excitation-emission matrix analysis. Recording of
FL emission signal for time resolved-FL, 3D-FL measurements and excitation-emission matrix
analysis was performed by TCSPC module. Slit widths were selected 5 nm and pathlength was 1
cm for all experiments and they were performed at 25oC. 2101-UV spectrophotometer (Shimadzu,
Japan) and INOVA 500 MHz spectrometer (Varian, USA) used for UV-Vis absorption and NMR
13
(1H and C) experiments. MALDI-TOF mass spectrometer (Bruker Daltonics Microflex, USA)
and Spectrum 100 spectrophotometer (Perkin Elmer, USA)were used for recording of mass and
FT-IR spectra of novel compounds. Non-linear regression analyses were applied by equations
using Sigma-Plot 14.0.
2.3. Synthesis of compounds
Synthesis and purification of 2-azido-1-ethanol, 9,10-bis(bromomethyl)anthracene, 2-(prop-2-
yn-1-yloxy)naphthalene were performed according to the literature [20]. N3-L and 1 which were
1
newly synthesized compounds were characterized by H, 13C NMR, FT-IR and MALDI-MS
6

spectra spectroscopies. All characterization results were consistent with structures of compounds
and they were given in Fig S1-S4.
2.3.1. 9,10-bis((2-azidoethoxy)methyl)anthracene (N3-L)
2-azido-1-ethanol (1.196 g, 13.73 mmol) and NaH (0.549 g, 13.73 mmol) was added in THF
(50 mL) under inert atmosphere and obtained suspension was refluxed for 30 min. After, 9,10-
bis(bromomethyl)anthracene (2.000 g, 5.490 mmol) which dissolved in THF (50 mL) was added
dropwise to suspension and reaction mixture was refluxed for 24 h. The reaction was followed by
TLC (silica gel used as stationary and 5 n-hexane: 1 ethylacetate (v/v) used as an eluent). After
reaction finished, G4 filter was used for filtration of reaction mixture and the organic solvent of
mixture was removed with using rotary evaporator under reduced pressure. Crude product was
purified by column chromatography (silica gel used as stationary and 5 n-hexane: 1 ethylacetate
(v/v) used as an eluent) and N3-L was obtained as light yellow solid (1.650 g, 80%, m.p. 92oC).
1H NMR (δppm, 298 K, DMSO-d6); 8.50 (dd, J = 6.9, 3.1 Hz, 2H), 7.61 (dd, J = 6.9, 3.1 Hz, 2H),
5.53 (s, 2H), 3.86 (t, J = 4.8, 2H), 3.47 (t, J = 4.8, 2H). 13C NMR (δppm, 298 K, DMSO-d6); 130.25,
130.01, 125.80, 124.10, 68.76, 64.30, 50.10. FT-IR (ATR, cm −1); 3094-3024 (-C=H), 2981– 2860
(-C-H), 2086.6 (-N=N=N), 1850-1650 (-C=C-), 1350-1250 (-C-H-). [M+H]+: 377.272 m/z (calc.
[M+H]+: 377.420).
2.3.2.
9,10-bis((2-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)methyl)
anthracene (1)
N3-L (0.200 g, 0.531 mmol), Cu(I)Br (0.229 g, 1.593 mmol), PMDTA (0.276 g, 1.593 mmol)
and 2-(prop-2-yn-1-yloxy)naphthalene (0.242 g, 1.328 mmol) were dissolved in dry
7

dichlorometane (10 mL) under inert atmosphere. The obtained solution was stirred at room
temperature for 24 h and followed by TLC (silica gel used as stationary and 2 THF: 1 n-hexane
(v/v) used as an eluent). After completion of the reaction, it was extracted with
dichloromethane/H2O. The organic phase was dried over Na2SO4 and rotary evaporator was used
to remove organic solvent. Crude product was purified by column chromatography (silica gel used
as stationary and 2 THF: 1 n-hexane (v/v) used as an eluent). Compund 1 was obtained as
white/light yellow solid (0.268 g, 68%, m.p. 186oC). 1H NMR (δppm, 298 K, CDCl3); 8.25 (dd, J =
6.6, 3.1 Hz, 4H), 7.73 (dt, J = 16.1, 7.7 Hz, 8H), 7.49 (dd, J = 6.7, 3.1 Hz, 4H), 7.43 (t, J = 7.4 Hz,
2H), 7.34 (t, J = 7.4 Hz, 2H), 7.21 (s, 2H), 7.11 (d, J = 10.2 Hz, 2H), 5.48 (s, 4H), 5.18 (s, 4H),
4.53 (t, J = 4.3 Hz, 4H), 4.04 (t, J = 4.3 Hz, 4H). 13C NMR (δppm, 298 K, CDCl3); 156.24, 134.39,
130.58, 129.63, 129.11, 127.64, 126.88, 126.42, 126.10, 124.60, 123.84, 118.82, 107.18, 68.58,
−1
64.40, 61.91, 37.48.FT-IR (ATR, cm ); 3074-3028 (-C=H), 2980– 2863 (-C-H), 1850-1650 (-
C=C-), 1350-1250 (-C-H-). [M]+: 740.901 m/z and [M+Na+K]+: 802.042 (calc. [M]+: 740.86 and
[M+Na+K]+: 802.86).
2.4. Real samples
Environmental water samples and medicine tablets were collected from Istanbul/Kocaeli,
Turkey. After filtration process of the samples by blue band filter paper, HNO3 (70%) was used
for the acidification of all samples. Before using for real sample analysis they stored at 4 oC in the
refrigerator. Medicine tablet samples were prepared for analysis according to the previous report
[21].
8

2.5. Measurement procedure
Sensitive and selective complexation of the presented FL sensor (1) with iron allowed
spectrofluorimetric analysis of iron level in real samples without any enrichment processes. The
FL emission of 1 in blue region which were originated from anthracene moiety significantly
quenched after complexation of 1 with iron. Because of HNO3 (70%) and other acidic medium of
digested and stored processes of real samples, Fe2+ ions of medicine tablets and environmental
water samples were oxidized to Fe3+. Therefore, complexation of 1 with Fe3+ was basis of
presented spectrofluorimetric determination method of iron. The determination of iron amount of
real samples was performed by calibration curve which was obtained with various amount of Fe3+
of water samples. Equation (1) was used for calculation of iron quantity.
(1)
where m, n, F and F0 are the slope of the calibration curve, intercept of FL emission signal axis,
FL emission signal of 1+Fe3+ and FL emission signal of 1 in the absence of Fe3+ (1:1 v/v, pH 8.0,
λex=345 nm, 2 μM 1), respectively. In addition, (F0-F)/F0 and CFe3+ are relative FL signal of 1+Fe3+
and iron concentration of real samples, respectively. The emission of anthracene moiety which
was the origin of the FL emission of sensor 1 was quenched with increased concentration of Fe3+.
This FL quenching was gradual and proportional until 130 µM of Fe3+ and it was applicable for
determination of iron real sample. Spectrofluorimetric analysis of iron with FL sensor 1 in
medicine tablets and environmental water samples was performed as follows; 5x10-4 M of 1 was
prepared in DMSO and it was used as stock solution. 0.200 mL of 1 was taken from stock solution
and added to 10 mL volumetric flask with 4.8 mL of DMSO. After, Britton–Robinson (B-R, 0.5
9

mL, pH 8.0) and real samples (0.5 mL) added to flask, the volume of flask was completed to 10
mL with deionized water. Finally, obtain solution was shaken carefully for 10 seconds. The
spectrofluorimetric analysis was performed according to relative FL response of 1+Fe3+ complex
which was recorded at 424 nm after optimization studies. The accuracy of the developed
spectrofluorimetric determination method was investigated by spike/recovery measurements and
inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis under optimum
analytical conditions.
2.6. Photophysical properties
The comparative method (Eq. (2)) was used for the calculation of the FL quantum yields (ΦF)
of 1 and 1+Fe3+ complex [22].
2
퐹.퐴 .푛
푆푡푑
∅퐹 = ∅퐹
(2)
푆푡푑
2
퐹
푆푡푑
.퐴.푛
푆푡푑
where n, F, FStd, A, and AStd are the refractive indices of solvents which were used for dissolution
of 1 and standard, FL peaks areas of FL sensor and standard, absorbance bands areas of FL sensor
and standard at same analytical conditions. In this study, we used quinine sulfate which has ΦF =
0.54 in 0.1 M H2SO4 as a standard [23].
The FL lifetimes of 1 and 1+Fe3+ complex was directly measured by time-correlated single-
photon counting (TCSPC) method with appropriate exponential calculations. All lifetime
measurements were performed with nanoLED (Horiba Jobin Yvon, France) at 390 nm as a light
source. The excitation–emission matrix (EEM) analysis and 3D-FL measurements were obtained
by CCD detector on Fluorolog 3-2iHR (Horiba Jobin Yvon, France) .
10

3. Results and discussion
3.1. Synthesis/structural characterization
Click chemistry has been extensively applied for metal determination studies with FL sensors.
Because 1,2,3 triazoles which are obtained as a result of Cu(I)-catalysed azide–alkyne
cycloaddition reaction can serve not only a bridge between ionophore and fluorophore groups but
also it can be used as ionophore [24]. Up to now, some fluorescent sensors for different metal ions
such as Pb2+, Ni2+, Cd2+, Zn2+, Ag+, Hg2+ and Fe2+/Fe3+ were synthesized via click chemistry to
form triazole groups as metal ions binding site, which were consisted from different alkoxy chain
lengths and chemical structures [9,24-27]. Apart from the chemical structure, alkoxy chain lengths
of triazole binding sites affect the sensitivity and selectivity of fluorescent sensors towards metal
ions which could be optimized via alkoxy chain lengths [24-26]. In recent years, due to its
important role in biological processes and industry, there are many fluorescent sensors that existed
in literature based on click chemistry and selective for iron [9,24,27]. With the experience that we
have gained from literature about chemical structures of click chemistry-based iron selective
fluorescent sensors, in this study, we synthesized di-podal anthracene-based FL sensor with click
reaction for spectrofluorimetric analysis of iron in real samples. For that purpose, synthesis and
characterization of 2-azido-1-ethanol, 9,10-bis(bromomethyl)anthracene, 2-(prop-2-yn-1-
yloxy)naphthalene were performed via reported procedures of the literature [20]. Then, as a result
of nucleophilic substitution reaction of 2-azido-1-ethanol and 9,10-bis(bromomethyl)anthracene
in the presence of NaH, N3-L which was azide-functionalized core compound was obtained.
Finally, di-podal anthracene-based FL sensor (1) was obtained by click reaction of N3-L with 2-
(prop-2-yn-1-yloxy)naphthalene (68% yield) (Scheme 1). Column chromatography was applied to
purify all novel compounds and MALDI-MS, 1H, 13C NMR and FT-IR spectroscopies were used
11

for chemical characterization of N3-L and 1 (Fig S1-S4). In this work, triazole groups are designed
as ionophores while naphthalene/anthracene groups are designed as fluorophore groups that
convert recognition into analytical signals.
Scheme 1.
1
As can be seen from H NMR spectra of N3-L and 1 (Fig S2 (a and b)), symmetric aromatic
anthracene peaks are observed in N3-L at 8.50 ppm and 7.61 ppm while at compound 1, triazole
and aromatic naphthalene peaks were observed with aromatic anthracene peaks between 8.26-7.10
ppm. In addition, integration values and chemical shifts of aromatic and methylene proton in the
1H NMR spectra of N3-L and 1 compatible with their structures.
According to FT-IR spectra of N3-L and 1 (Fig S4), H-C≡C- stretching vibrations of 2-(prop-
2-yn-1-yloxy)naphthalene observed at 3293 cm-1. In addition, obtained -N=N=N (azide) vibration
which was observed at 2086 cm-1 after nucleophilic substitution reaction of 2-azido-1-ethanol with
9,10-bis(bromomethyl)anthracene confirmed chemical structure of N3-L in the FT-IR spectrum.
After click reaction of N3-L with 2-(prop-2-yn-1-yloxy)naphthalene to obtain FL sensor 1, H-
C≡C- stretching vibrations of 2-(prop-2-yn-1-yloxy)naphthalene and N=N=N (azide) vibration of
N3-L were disappeared. -CH and -C=H vibrations existed in FT-IR spectrum which supported the
formation of FL sensor 1 (Fig S4).
3.2. Spectroscopic studies of compound 1
The absorption and FL properties of 1 were studied in various solvents including n-hexane,
THF, dichloromethane, ACN, ethanol, DMF, DMSO, water, and DMSO: water (1:1) with different
concentrations via Ultraviolet-Visible (UV-Vis) absorption and FL spectrophotometer at 25 °C.
12

According to Fig. S5, absorption properties of 1 do not differ by changing the solvent system and
compound 1 insoluble in n-hexane. The difference in the absorbance intensity is probably related
to the difference in the dissolution of compound 1 in various solvents. Since compound 1 contains
both naphthalene and anthracene parts, its UV-Vis spectrum has two electronic absorption maxima
which are located at wavelengths of 274 nm and 366 nm and they are ascribed to π–π* transition
[24]. Also, the same solvents were used to investigate the FL emission of 1. According to the Fig
S6, severe FL emission responses are perceived at about 410 nm and 424 nm (λex= 345 nm) for
prepared compound 1 (except in water), which is consistent with reported results for FL emission
of anthracene. According to the results of Fig S5 and S6, the solvents systems do not affect so the
optical properties of 1, except in more polar solvents such as water the excimer emission vanishes
which is related to the π- stacking interactions disrupting [9]. Aqueous DMSO system (1:1 v/v)
gives suitable FL signals at 410 nm and 424 nm with a 50 nm Stokes shift for spectrofluorimetric
detection of Fe3+ in environmental water samples (Fig S7).
3.3. Optimization of compound 1 procedures
In order to get high sensitivity and selectivity of the compound 1 complexation procedure for
Fe3+ detection, the reaction conditions were optimized. The effects of the influential parameters
including competitive ions, initial 1 concentration, pH, time before measurement, and buffer
concentration were investigated.
3.3.1. Effect of competitive metal ions
The most important parameter in FL sensors is its selectivity which becomes even more
significant for analysis of real samples. UV-Vis absorption and FL spectroscopies were used for
13

studying the selectivity of 1. All spectral determinations were carried out in spectroscopic quartz
cuvette with final solution volume of 10 mL via applying micropipette at room temperature. The
stock solutions of chloride salts of cations (except for Pb2+ and Ag+) were prepared in water whilst
the stock solution of 1 was prepared in DMSO. UV-Vis and FL characteristics of compound 1 in
buffered aqueous DMSO solution (1:1 v/v and pH 8.0) were evaluated by addition of 130 µM
various competitive metal ions like Fe3+, Fe2+, Zn2+, Pb2+, Ni2+, Na+, Mn2+, Mg2+, Li+, Hg2+, K+,
Cu2+, Cd2+, Ca2+, Cs+, Co2+, Cr2+, Ba2+, Al3+, and Ag+ when excited at 345 nm. According to Fig
1a, addition of Fe2+/Fe3+ to 10 µM sensor 1 causes significantly increase (about 4 fold) in UV-Vis
absorption whereas other different metal ions addition does not affect absorption spectra. These
increases in UV-Vis absorption spectra of compound 1 attributed to the formation of compound
1-Fe3+/2+ complexes, which causes the electron reorganization with adding of Fe3+/2+ [28]. Also,
significant color change is observed by adding Fe3+/Fe2+ to aqueous DMSO solution of 1 while
other different metal cations don’t make remarkable changes in solution color at daylight (Fig 1c).
In order to investigate the selectivity of the sensor, the FL of 2 µM compound 1 was also evaluated
in the presence of competitive metal cations with the identical operation conditions with UV-Vis
electronic absorption spectroscopy. As it can be observed at Fig 1b and 1d, by adding Fe3+/Fe2+ to
the sensor 1, its FL emission signal is fully quenched, along with FL colure change from colorless
to yellow. The FL quenching is generally explained via chelation enhanced fluorescent quenching
(CHEQ) mechanism in which coordination of paramagnetic guests like Fe3+ and Fe2+ with the host
causes the quenching of the fluorophore FL emission [29].
Fig 1.
14

As shown at Fig 2, the relative FL intensity changes of compound 1 in the presence of cations
(Fe3+, Fe2+, Zn2+, Pb2+, Ni2+, Na+, Mn2+, Mg2+, Li+, Hg2+, K+, Cu2+, Cd2+, Ca2+, Cs+, Co2+, Cr2+,
2-
-
-
-
2-
-
-
Ba2+, Al3+, and Ag+), anions (SO4 , CN-, F-, I-, H2PO4 , HSO4 , NO3 , CO3 , Cl-, S2O3 , NO2 , S2-,
CH3COO-, SCN-) and single molecules (ascorbic acid, catechin, rutin, quercetin, citric acid, caffeic
acid and oleic acid) confirms the selectivity of the sensor towards Fe3+/Fe2+ detection. The
difference of proposed sensor 1 relative FL intensity between iron-free metal blend and iron-
containing metal blend demonstrates that different competitive ions do not affect the relative FL
intensity of 1.
Fig 2.
3.3.2. Effect of pH
Due to the efficacy of pH on the chemical structure of 1 and its complexation with Fe3+, pH of
the solution is one of the main parameters for quantitative recovery. Therefore, the effect of various
pH values (4.0-10.0) with Britton-Robinson (B-R) buffer system on relative FL responses of
1+Fe3+ was examined while other operational conditions were kept constant. Maximum relative
FL emission intensity is observed at pH 8.0 and it decreases below and above 8.0 (Fig S8a). Above
pH 6, compound 1 acts mainly as an electron donor, thus relative FL emission signal increases
significantly [30]. Since the formation of complex, sensitivity, and selectivity were considered,
Britton-Robinson buffer with pH 8.0 was selected for determination process of environmental
water samples.
15

3.3.3. Effect of the B-R buffer concentration
Determination of appropriate buffer with proposed medium pH is needed to attain stable Fe3+-
compound 1 complexes [30]. Therefore, the evaluation of efficacy of the B-R buffer solution’s
quantity on the interaction of 1 with Fe3+ should be performed. In order to this purpose, the effect
of B-R buffer solutions with different concentrations from 0.1 M to 0.4 M on relative FL emission
signal of the complex was studied. As demonstrated in Fig S8b, by increasing the concentration of
the B-R buffer above 0.1 M, the relative FL intensity of Fe3+- compound 1 decreases. So, 0.1 M
B-R buffer solution is suitable for complexation process.
3.3.4. Effect of initial 1 concentration
Initial 1 concentration is another parameter which should be optimized to get more sensitivity.
For this aim, different concentrations of 1 ranging from 1 µM to 4 µM were prepared in aqueous
DMSO solution (1:1 v/v). The differences in the intensity of relative FL by adding Fe3+ have been
investigated in Fig S8c. When 1 concentration changes from 1 µM to 2 µM, the relative FL
intensity increases. But by following the concentration increasing up to 4 µM, the intensity of
relative FL decreases sharply. This behavior could be attributed to intermolecular self-quenching
of anthracene groups in high concentrations that have been observed for several fluorophores [31].
According to obtained results, 2 µM was chosen as sufficient 1 concentration for the Fe3+ detection
process in real samples.
3.3.5. Effect of measurement time
The time before determination is a parameter that affects the complexation of 1 with Fe3+. So,
its effect on Fe3+ detection process was evaluated in the range of 0-50 second (s) while other
16

parameters kept constant. As illustrated in Fig S8d, when 2 µM of 1 in buffered aqueous DMSO
solution (1:1 v/v, pH 8.0) is used, the maximum intensity of relative FL is obtained at 10 s and
then increasing the time doesn’t show any effect on FL intensity. Therefore, the determination
process carried out after 10 s in real samples, considering the stability of 1+Fe3+ complexes.
3.4. Formation of 1-Fe3+ complexes and quenching mechanism of 1 by Fe3+
In order to study the formation of 1- Fe3+ complexes, Job’s (continuous variation) plot and also
non-linear curve fitting analyses were carried out in water:DMSO (1:1 v/v, pH 8.0) [32]. Job’s
(continuous variation) plot was obtained by using 2 μM compound 1 and Fe3+ ions mole fraction
increasing in complexes (Fig S9a). The maximum FL intensity of complexes attains at 0.5, it can
be concluded that 1+Fe3+ complexes stoichiometry is 1:1 (metal:ligand). As can be observed at
non-linear curve fitting analysis (Fig S9b), by addition of Fe3+ to compound 1, relative FL intensity
increases and an inflection point is demonstrated at plot where 1:1 (metal:ligand) stoichiometry
was determined for compound 1 to Fe3+. The results obtained from non-linear curve fitting plots
and Job’s plot analysis are in compatibility with each other.
In FL sensor applications, the photostability of receptors and complexes is necessary to obtain
highly accurate and precise results. 2 µM compound 1 containing 130 µM Fe3+ in buffered aqueous
DMSO solution (1:1 v/v, pH 8.0) was used to appraise photostability of 1. Then, the FL
measurements were performed between 0 and 60 minutes in daylight. As can be seen at Fig S10,
compound 1 and its complex with Fe3+ both are highly photostable and their relative FL intensities
are unaffected and relatively constant during 60 min. In order to appraise the reversibility of the
compound 1 reversibility towards Fe3+, fluoride ions (F-) were added to Fe3+-1 complexes.
Restitution of the increased UV-Vis absorption and quenched FL intensity of compound 1 by
17

adding Fe3+ after addition of different concentrations of F- demonstrates that identification of Fe3+
is a complexing and reversible process (Fig 3 (a-d)).
Fig 3.
FL quenching process occurs in two different ways: dynamic and static processes. Static
quenching is caused by nonfluorescent complex, when a quencher and fluorophore complexes in
ground state. Encounter of quencher and excited state of fluorophore molecules causes dynamic
quenching process [33]. So, FL lifetime of the fluorophore is imported in dynamic process which
must be reduced while it does not affect the static process. The FL emission intensity is also
affected by the quencher concentration which can shed light into the quenching mechanism. A
quencher’s performance in the quenching mechanisms is given by Stern-Volmer equation (Eq. (3))
[34]:
I0
(3)
 Ksv

Q 1

I
where, I0, I, Ksv, and Q represent the initial FL intensity of a fluorophore (1), final FL signal of a
fluorophore in the quencher presence (Fe3+), Stern-Volmer constant, and quencher’s concentration
(molar). According to Stern-Volmer relationship, in static quenching a linear graph obtains from
plotting of I0/I against [Q] with y-axis intercept of 1. Positive deviation observation in the graph
by increasing quencher concentration demonstrates that the effective quenching mechanisms in
the system are both dynamic and static quenching [24]. Stern-Volmer plot of Fe3+-1 complex is
demonstrated at Fig S11a. As the results show, by increasing Fe3+ concentration until 9×10-5 M,
18

the I0/I increase linearly and further the positive deviations are perceived for Fe3+-1. So, it can be
understood that FL intensity quenching process of compound 1 is included both dynamic and static
quenching. As pointed before, an alternative way to recognize quenching process is FL lifetimes.
So, the FL lifetime determinations of compound 1 and in the presence of Fe3+ and F- were carried
out (Fig S11b). The FL lifetimes were calculated as 9.135±0.012 ns (τ0), 8.867±0.015 ns (τ1),
8.951±0.017 ns (τ2) for compound 1, compound 1+Fe3+, and compound 1+Fe3++F-, respectively.
Owing to the formation of Fe3+-1 complex in ground state, slight differences might be seen in the
FL lifetimes of free sensor and complex. When the Fe3+-1 complex forms, sensor 1 becomes non-
fluorescent and in the FL lifetime determination, the static quenching causes deletion of this non-
fluorescent part. Therefore, the FL lifetime is due to non-complexed compound 1 and the FL
properties of free part of compound 1 remain initial. So, it is expected that in static quenching
process τ0/τ to be ~1 [33,9]. The rate of the FL lifetimes of 1 (τ0) and its complex with Fe3+ (τ) was
calculated as 1.03. According to results, it seems that the effective mechanism for 1+Fe3+
complexation is static quenching.
3.5. Analytical figures of sensor 1
FL titration experiments were performed by 2 µM of compound 1 and different concentrations
of Fe3+ (0-130 µM) and F- (0-450 µM) with the aim of determination of dynamic range of the
sensor. As shown at Fig 3c and 3d, when Fe3+ concentration increases gradually, relative FL
emission intensity proportionally quenches at 424 nm and by adding F- to the sensor, the FL
intensity increases again. Fig 4 demonstrates the calibration curves of 1-Fe3+ and 1-Fe3++F-.
According to the calibration curves, the linear ranges of the sensor are between 0.3-130 µM of
-
Fe3+ ((F0-F)/F0 = 0.0068CFe3+ + 0.054, R2=0.9973) and 0.3-450 µM of F- ((F0-F)/F0 = -0.002CF +
19

0.9683, R2=0.9945) with limit of detection (LOD) of 0.314 µM, which is calculated with using
3σ/k (where k: the calibration curve’s slope, σ: the blank solution’s deviation). The limit of
quantification (LOQ) was also computed as 0.942 µM for Fe3+ detection. Importantly, obtained
LOD and LOQ values for determination of Fe3+ with presented spectrofluorimetric method is
lower than the maximum EPA’s permitted level of Fe3+ ions in drinking water (5.4 µM or 0.3 ppm)
[35]. Another important parameter for consistent results is precision. Under optimized conditions,
the calculated relative standard deviation (RSD%) for Fe3+ was 2.89% (N=10). Table S1 displays
the analytical performance of 1, which the high sensitivity, selectivity, and reproductively of the
sensor can be concluded.
Fig 4.
3.6. Compound 1 excitation–emission matrices (EEMs) changes during Fe3+ detection
process
Comparative content of the fluorescent components is the information obtained from EEM
spectra [36]. Target analytes’ composition is provided from peak position and their red or blue
shifts is related to their surroundings and sources [37]. Unlike UV-Vis, intrinsic FL is comprising
data about dynamic properties due to intermolecular and intramolecular interactions, as well as
substances’ heterogeneity, conformation, structure, and functional groups [38]. According to Fig
5, compound 1 is a fluorescent substance and the presence of Fe3+ and F- is effective on its FL
spectra. 1 has an emission peak which is located at 400-500 nm and indicates its blue emission
(Fig 5a, S12a). On the other hand, addition of Fe3+ quenches the FL intensity of 1, which illustrates
20

the interaction of Fe3+ with compound 1 (Fig 5b, S12b). Further, when F- is augmented 1+Fe3+
complex separates and FL intensity of 1 restore (Fig 5c, S12c).
Fig 5.
3.7. Analysis of real samples
The performance of the method for analysis of real samples through Fe3+ measurement was
appraised in environmental water samples and medicine tablets. For this purpose, spike and
recovery tests and also official ICP-OES method were performed. Environmental water and
medicine tablet samples were spiked between 0 and 75 µM Fe3+ and the purposed FL method was
exerted under optimum conditions. As demonstrated in Table 1, the obtained quantitative
recoveries with low RSD% are in good agreement with spiked amounts of analyte. ICP-OES was
used as an official method in order to more verification of the pointed FL method accuracy (Table
2) and the evaluation of the differences between 2 methods was carried out by the Student t-test,
which the results can be seen at Table S2. According to the results, the differences were found to
be insignificant. The obtained results including RSD%, recoveries, and t-test demonstrate the high
precision and accuracy of the presented FL method for the determination of Fe3+ in tablet and
water samples. The presented FL method was compared with some other analytical methods such
as potentiometry, spectrophotometry and spectrofluorimetry for determination of iron content of
real samples and results were given at Table S3. According to Table S3, presented FL method has
higher sensitivity, wider linear range and importantly lower limit of detection than given
potentiometric, spectrophotometric and spectrofluorimetric methods, without interfering [S1-9].
The simple and efficient spectrofluorimetric determination of iron in real samples can performed
21

at natural pH in several seconds due to sensitive and selective interaction of 1 with iron. Although
some determination methods which based on spectrophotometry has lower detection limit than
presented method, enhancement procedures using eluent and adsorbent are applied before
determination via these methods [S10]. The results of comparison revealed that, presented
spectrofluorimetric method is simple, selective, time-efficient, accessible and sensitive
determination method for iron in real samples.
Table 1
Table 2
4. Conclusion
A novel dipodal receptor based on anthracene was introduced with the quenching effect of iron
ions, which could be used as a sensor for Fe3+ detection. The synthesis of this turn-off fluorescent
sensor was performed by the proposed synthetic strategy. FL spectroscopy and UV–Vis absorption
demonstrated the sensor’s high sensitivity and selectivity towards iron ions at low levels. ICP-OES
analysis, spike and recovery tests were carried out to validate the introduced sensor, which showed
the high accuracy of the sensor. This approach with LOD of 0.314 µM in the linear range of 0.3-
130 µM could be appropriate for spectrofluorimetric analysis in biological and water samples for
quantification of iron level. More importantly, the proposed strategy could be used for obtaining
novel sensors for many various metal ions using a diversity of fluorophores and ionophores.
Author contributions
22

Alireza Khataee acted as a supervisor. Material preparation, data collection and analysis were
performed by Süreyya Oğuz Tümay. The first draft of the manuscript was written by Mahsa
Haddad Irani-nezhad and all authors commented on previous versions of the manuscript. All
authors read and approved the final manuscript.
Electronic supplementary material
The online version of this article contains supplementary material, which is available to
authorized users.
Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Gebze Technical University and University of Tabriz for the support provided.
23

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