Development of a fluorescent chemosensor for chloride ion detection in sweat using Ag+-benzimidazole complexes

Article abstract of DOI:10.1016/j.dyepig.2020.108291

The level of chloride ions (Cl?) in sweat is a recognized biomarker for the genetic disorder cystic fibrosis (CF). The accurate quantitation of chloride ions in sweat is therefore a vital diagnostic tool for this life-threatening disease. In this work, a fluorescent, chloride ion chemosensor was developed by exploiting the strong interaction between silver (Ag⁺) and chloride ions. Using the concept of ligand displacement, six Ag⁺-benzimidazole complexes were prepared as candidate chloride ion chemosensors. The Ag⁺-2-(Furan-2-yl)-1H-benzo[d]imidazole (Ag⁺-FBI) complex was identified as the optimal sensor owing to its Ag⁺-FBI high sensitivity (limit of detection = 19 μM), short response time (<3 min), and remarkable fluorescence turn-on response across a broad pH range (pH 6–9). The Ag⁺-FBI complex exhibited high selectivity for Cl? ions and was successfully used to quantify chloride ions in artificial sweat samples containing multiple ions and other biological constituents. This unique, simple, and effective probe thus shows great potential for clinical diagnostic applications.

Full text of DOI:10.1016/j.dyepig.2020.108291

Journal Pre-proof
Development of a fluorescent chemosensor for chloride ion detection in sweat using
+
Ag -benzimidazole complexes
Jaewon Kim, Suji Lee, Sudeok Kim, Minhyuk Jung, Hohjai Lee, Min Su Han
PII:
S0143-7208(19)32715-9
DOI:
https://doi.org/10.1016/j.dyepig.2020.108291
DYPI 108291
Reference:
To appear in: Dyes and Pigments
Received Date: 25 November 2019
Revised Date: 18 February 2020
Accepted Date: 18 February 2020
Please cite this article as: Kim J, Lee S, Kim S, Jung M, Lee H, Han MS, Development of a fluorescent
+
chemosensor for chloride ion detection in sweat using Ag -benzimidazole complexes, Dyes and
Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108291.
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2020 Published by Elsevier Ltd.

CRediT author statement
Title: Development of a fluorescent chemosensor for chloride ion detection in sweat
+
using Ag -benzimidazole complexes
-
Jaewon Kim: Methodology, Investigation, Visualization, Writing – Original
Draft
-
-
-
-
-
Suji Lee: Methodology, Visualization, Writing – Original Draft
Sudeok Kim: Methodology
Minhyuk Jung: Investigation
Hohjai Lee: Project administration
Min Su Han: Conceptualization, Writing – Review & Editing, Project
administration

Graphical Abstract

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Development of a fluorescent chemosensor for chloride ion detection in
+
sweat using Ag -benzimidazole complexes
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‡
‡
Jaewon Kim , Suji Lee , Sudeok Kim, Minhyuk Jung, Hohjai Lee* and Min Su Han*
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Department of Chemistry, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu,
Gwangju 61005, Republic of Korea
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*Corresponding authors. hohjai@gist.ac.kr (H. Lee), happyhan@gist.ac.kr (M. S. Han)
‡
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J. K. and S. L. contributed equally to this work.
Abstract
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The level of chloride ions (Cl ) in sweat is a recognized biomarker for the genetic disorder cystic fibrosis (CF).
⁻
The accurate quantitation of chloride ions in sweat is therefore a vital diagnostic tool for this life-threatening
disease. In this work, a fluorescent, chloride ion chemosensor was developed by exploiting the strong interaction
+
+
between silver (Ag ) and chloride ions. Using the concept of ligand displacement, six Ag -benzimidazole
+
complexes were prepared as candidate chloride ion chemosensors. The Ag -2-(Furan-2-yl)-1H-
+
+
benzo[d]imidazole (Ag -FBI) complex was identified as the optimal sensor owing to its Ag -FBI high
sensitivity (limit of detection = 19 µM), short response time (< 3 min), and remarkable fluorescence turn-on
+
response across a broad pH range (pH 6-9). The Ag -FBI complex exhibited high selectivity for Cl
⁻
ions and
was successfully used to quantify chloride ions in artificial sweat samples containing multiple ions and other
biological constituents. This unique, simple, and effective probe thus shows great potential for clinical
diagnostic applications.
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Keywords: Metal-ligand exchange; Ligand displacement; Chloride ion; Fluorescent chemosensor; Cystic
fibrosis; Sweat test
i

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1. Introduction
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Chloride ions (Cl ) are the most abundant ions in biosystems [1]. They are ubiquitous in agricultural,
environmental, industrial, and physiological systems and are extensively used in organic chemicals, agricultural
fertilizers, and even as food additives [2-4]. In clinical settings, chloride ions in sweat are used as a biomarker
for the genetic disorder cystic fibrosis (CF), which is caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene [5-9]. The CFTR protein functions as a chloride ion channel and abnormal
proteins result in increased levels of chloride ions being present in sweat, making the detection and
quantification of sweat chloride an invaluable tool for diagnosing CF [10]. Although several analytical
techniques exist for detecting and quantifying chloride ions, including ion chromatography and electroanalytical
chemistry methods involving potentiometry and coulometry, they are limited by the accessibility and complexity
of the instrumentation [11-16]. A potential alternative to these systems is chemosensors, which benefit from both
ease of use and faster detection. Unfortunately, despite the development of multiple chloride ion chemosensors
over the past two decades, few have been applicable for the analysis of biological samples owing to the lack of a
specific recognition unit for the chloride ion [17-30].
+
-10
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Silver ions (Ag ) strongly bind to chloride ions (K (AgCl) = 1.77 × 10 ), making them a valuable
sp
reagent for the specific recognition of chloride ions [31]. The strong interaction between silver and chloride ions
was exploited for the development of the established Mohr method for chloride ion detection. This technique is
based on precipitation titration of AgCl using silver nitrate (AgNO ) as the titrant and potassium chromate
3
(K CrO ) as the indicator [32,33]. While this method is still widely used to analyze halide ions, it suffers from
2
4
requirements of large reagent volumes and alkaline working conditions. Despite the limitations of the Mohr
method, the strong interaction between silver and chloride ions presents a clear opportunity for the development
of a sensitive and selective chemosensor for chloride ion detection.
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A great number of chemosensors have been developed using the concept of ligand displacement [34-
41]. This design relies on the change in the optical properties of a free and metal-complexed fluorescent or
chromogenic dye. Analyte detection is based on the ligand exchange reaction between the metal-dye complex
and an analyte with a strong binding affinity for the complexed metal ion. Herein, we developed a selective,
+
fluorescent chemosensor for chloride ions based on ligand displacement from Ag -benzimidazole complexes.
Six benzimidazole derivatives (1a-1c, 2a-2c) were synthesized as fluorescent ligands based on previous reports
1

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describing N-heterocycles, and particularly benzimidazoles, as good ligands for silver ions [42-46].
+
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Fluorescence-quenched Ag -benzimidazole complexes (Ag -1a-1c, 2a-2c) were prepared and investigated to
determine their ability to detect chloride ions via ligand exchange-induced fluorescence recovery (Scheme 1).
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Scheme 1. Chloride ion chemosensor based on ligand displacement from Ag -benzimidazole complexes.
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2. Experimental
2.1. Materials and instrumentation
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Chemical reagents were purchased from commercial sources (Sigma-Aldrich, Alfa Aesar, Duksan,
1
Daejung, and Tokyo Chemical Industry) and, unless otherwise stated, were used without further purification. H-
1
3
and C-NMR spectra were recorded on a JEOL 400 MHz NMR spectrometer. Mass spectra were obtained using
an Agilent ESI-Q/TOF (quadrupole/time-of-flight) mass spectrometer. Melting points were measured using a
BUCHI Melting Point M-565. FT-IR spectra were obtained using a Thermo Scientific Nicolet iS10 FT-IR
TM
spectrometer. UV-Vis and preliminary fluorescence spectra were recorded on a BioTek Cytation 3 Cell
Imaging Multi-Mode Reader. Fluorescence spectra were obtained using an Agilent Cary Eclipse fluorescence
spectrophotometer.
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2.2. General synthesis and characterization
2.2.1. General procedure for the synthesis of 2-substituted benzimidazoles [47]
7
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Scheme 2. General synthesis of benzimidazole derivatives (1a-1c, 2a-2c).
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A mixture of aldehyde (10.00 mmol) and NaHSO (11.0 equiv., 11.45 g) in H O (30 mL) was heated
3 2
to reflux. A solution of an o-phenylenediamine derivative (10.00 mmol) in H O (10 mL) was added dropwise to
2
the solution and the reaction was further refluxed. Upon completion, the reaction mixture was cooled to room
temperature and the precipitate was collected by vacuum filtration. The residue was washed with H O and dried
2
under vacuum.
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2.2.2. Characterization of 2-substituted benzimidazoles
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2-(Thiophen-2-yl)-1H-benzo[d]imidazole (1a) [47,48] (1.67 g, 84%) pale yellow solid. mp 327-332 ℃ ; H-NMR
(400 MHz, DMSO-d ) δ 12.95 (s, 1H), 7.84 (dd, J = 3.7, 1.2 Hz, 1H), 7.72 (dd, J = 5.2, 1.1 Hz, 1H), 7.56 (q, J =
6
1
3
3.0 Hz, 2H), 7.24-7.17 (m, 3H); C-NMR (100 MHz, DMSO-d ) δ 147.6, 139.7, 134.2, 129.3, 128.8, 127.3,
6
122.7, 115.4.
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3
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2-(Thiophen-2-yl)-1H-benzo[d]imidazole-6-carbonitrile (1b) (0.35 g, 42%) pale yellow solid. mp 245-248 ℃ ;
1
H-NMR (400 MHz, DMSO-d ) δ 13.47 (s, 1H), 8.08 (s, 1H), 7.92 (d, J = 4.5 Hz, 1H), 7.79 (dd, J = 5.0, 1.0 Hz,
6
13
1H), 7.68 (d, J = 8.3 Hz, 1H), 7.56 (dd, J = 8.4, 1.5 Hz, 1H), 7.24 (dd, J = 5.0, 3.8 Hz, 1H); C-NMR (100 MHz,
8
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7
DMSO-d , at 90 ℃) δ 150.2, 141.9, 139.6, 132.7, 129.9, 128.4, 128.2, 125.7, 120.1, 119.8, 115.6, 104.3; ESI-
6
HRMS: m/z calcd for C H N S + H: 226.0433, found 226.0434.
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6-Methoxy-2-(thiophen-2-yl)-1H-benzo[d]imidazole (1c) (1.15 g, 50%) pale yellow solid. mp 70-73 ℃
1
(decomposed); H-NMR (400 MHz, DMSO-d ) δ 12.80 (s, 1H), 7.78 (d, J = 3.8 Hz, 1H), 7.68 (d, J = 5.3 Hz,
6
1H), 7.45 (d, J = 5.3 Hz, 1H), 7.21 (q, J = 2.8 Hz, 1H), 7.05 (s, 1H), 6.83 (dd, J = 9.2, 2.3 Hz, 1H), 3.80 (s, 3H);
1
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C-NMR (100 MHz, DMSO-d ) δ 156.4, 134.5, 128.7, 126.6, 112.0, 56.0; ESI-HRMS: m/z calcd for
6
C H N OS + H: 231.0587, found 231.0593.
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2-(Furan-2-yl)-1H-benzo[d]imidazole (2a, FBI) [47,48] (1.8236 g, 99%) brown solid. mp 271-273 ℃; H-NMR
(400 MHz, DMSO-d ) δ 12.99 (s, 1H), 7.96 (q, J = 0.8 Hz, 1H), 7.56 (q, J = 3.1 Hz, 2H), 7.23-7.19 (m, 3H),
6
1
3
6.74-6.72 (m, 1H); C-NMR (100 MHz, DMSO-d ) δ 145.8, 145.3, 144.0, 139.2, 122.9, 115.5, 112.9, 111.3.
6
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2-(Furan-2-yl)-1H-benzo[d]imidazole-6-carbonitrile (2b) (1.67 g, 80%) pale brown solid. mp 226-229 ℃; H-
NMR (400 MHz, DMSO-d ) δ 13.46 (s, 1H), 8.07 (s, 1H), 7.99 (q, J = 0.8 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H),
6
13
7.56 (dd, J = 8.5, 1.5 Hz, 1H), 7.31 (dd, J = 3.4, 0.6 Hz, 1H), 6.74 (q, J = 1.7 Hz, 1H); C-NMR (100 MHz,
DMSO-d , at 90 ℃) δ 146.6, 145.4, 144.9, 141.6, 139.5, 125.7, 120.3, 119.8, 115.8, 112.5, 112.2, 104.4; ESI-
6
3

1
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HRMS: m/z calcd for C H N O + H: 210.0662, found 210.0664.
12 7 3
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2-(Furan-2-yl)-6-methoxy-1H-benzo[d]imidazole (2c) (0.62 g, 29%) pale brown solid. mp 142-145 ℃
1
(decomposed); H-NMR (400 MHz, DMSO-d ) δ 12.78 (s, 1H), 7.91 (q, J = 0.8 Hz, 1H), 7.45 (d, J = 7.9 Hz,
6
1H), 7.13 (d, J = 4.0 Hz, 1H), 6.98 (s, 1H), 6.83 (dd, J = 8.9, 2.4 Hz, 1H), 6.70 (q, J = 1.7 Hz, 1H), 3.79 (s, 3H);
1
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C-NMR (100 MHz, DMSO-d ) δ 170.9, 156.5, 146.2, 144.8, 112.8, 112.3, 110.3, 55.9; ESI-HRMS: m/z calcd
6
for C H N O + H: 215.0815, found 215.0819.
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2.3. Fluorescence-based screening of Ag -benzimidazole complexes
2.3.1. Identification of excitation and emission wavelengths (λ , λ ) of candidate fluorophores
ex em
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09
Solutions of 1a-1c and 2a-2c in DMSO (25 µM, 10% DMSO) were added to pH 8.0 HEPES buffer
(20 mM). Excitation and emission spectra of all candidates were recorded using a microplate reader.
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2.3.2. Fluorescence-based screening of benzimidazole derivatives as ligands for silver ions
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Solutions of benzimidazole derivatives 1a-1c and 2a-2c in DMSO (25 µM, 10% DMSO) were added
+
to pH 8.0 HEPES buffer (20 mM). Ag solutions (0-2.5 mM) in deionized distilled water were then added to
give a final sample volume of 200 µL and 10% DMSO. The samples were mixed, incubated for 30 min at room
temperature, and used for fluorescence intensity measurements. Fluorescence spectra were obtained using the
λ measured in section 2.3.1. (1a: λ = 315 nm, 1b: λ = 323 nm, 1c: λ = 316 nm, 2a: λ = 307 nm, 2b: λ =
ex
ex
ex
ex
ex
ex
315 nm, 2c: λ = 323 nm).
ex
+
-
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2.3.3. Fluorescence-based screening of Ag -dye complexes as Cl probes
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Solutions of benzimidazole derivatives 1a-1c and 2a-2c in DMSO (25 µM, 10% DMSO) were added
+
to pH 8.0 HEPES buffer (20 mM). Ag (125 µM) in deionized distilled water was added to the samples to give a
final volume of 200 µL and 10% DMSO. The samples were then mixed and incubated for 30 min at room
temperature. Solutions of Cl
⁻
(0-1 mM) in deionized distilled water were added to the samples, which were
then mixed, incubated for 10 min at room temperature, and used for fluorescence intensity measurements.
Fluorescence spectra were obtained using the λ measured in section 2.3.1. (1a: λ = 315 nm, 1b: λ = 323 nm,
ex
ex
ex
1c: λ = 316 nm, 2a: λ = 307 nm, 2b: λ = 315 nm, 2c: λ = 323 nm).
ex
ex
ex
ex
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2.4. Optimization of conditions for preparing the Ag -FBI complex
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2.4.1. Preparation of Ag -FBI complexes and fluorescence analysis
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A solution of FBI in DMSO (25 µM) was adjusted to a pH of 7.4 using HEPES buffer (pH 7.4, 20
+
mM). Individual samples of the Ag -FBI complexes were prepared by adding AgNO solutions (0-1 mM) in
3
+
deionized distilled water to give a final sample volume of 1 mL and 10% DMSO. The Ag -FBI complexes were
+
then incubated for 30 min at room temperature. These conditions were employed for the preparation of the Ag -
FBI complex across all the experiments with only the concentration of the AgNO solution being varied.
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33
Fluorescence spectra (λex = 307 nm) were recorded in triplicate, normalized, and the average value
and error were calculated. This procedure was used for the remainder of the experiments unless otherwise stated.
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2.4.2. Job’s method (Job plot) [49,50]
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To determine the binding stoichiometry, the binding mode of the Ag -FBI complex was investigated
using the method of continuous variation (Job’s method). The Job plot was obtained by plotting the difference in
the fluorescence intensity at 344 nm (∆F.I. = (F – F) × [FBI]) against the mole fraction of the probe
0
+
+
[Ag ]/([FBI] + [Ag ]). The resulting curve was fitted with non-linear curve parameters using OriginPro 2018b
software.
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2.4.3. Structural determination of the Ag -FBI complex using FT-IR and NMR studies
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42
For FT-IR analysis, Ag -FBI complex samples were prepared as previously described using different
equivalents of AgNO (0, 0.5, 1, 3) and then lyophilized. FT-IR spectra were obtained using KBr pellets.
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44
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For NMR analysis, Ag -FBI complex samples were prepared by mixing FBI (100 mM) in DMSO-d
6
with different equivalents of AgNO (0, 0.5, 1, 2, 3, 4, 5) in DMSO-d and incubating for 30 min at room
3
6
temperature.
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47
2.5. Study of Ag -FBI complex as a fluorescent chemosensor of Cl
⁻
ions
2.5.1. Effect of Cl
⁻
ions on fluorescence
+
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1
1
48
49
50
51
The Ag -FBI complex was prepared as described in section 2.4.1. using a 125 µM AgNO solution.
3
The mixture was stirred, incubated for 30 min at room temperature, and Cl
⁻
solutions (0-10 mM) in deionized
distilled water were added. The chloride ion-containing solutions were mixed and incubated for an additional 10
min at room temperature. The final sample preparation, spectral measurements, and data work-up were
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53
performed as previously described.
2.5.2. Fluorescence response of chloride ion-ligand exchanged solutions at various pH
+
1
1
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55
Ag -FBI samples were prepared as described in section 2.4.1. After adjusting the pH (pH range of
4.0–9.0), fluorescence spectra (λ = 307 nm) were recorded.
ex
1
1
1
1
56
57
58
59
Samples were prepared as described in section 2.5.1. using a 1 mM Cl solution. Measurements and
⁻
data analysis were performed as previously described. The pH of the final samples was adjusted to between 4.0
and 9.0. Fluorescence spectra were used to examine the effect of ligand exchange on the fluorescence of the
+
Ag -FBI complex.
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60
2.5.3. Ionic constituent selectivity tests
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Sample preparation, measurements, and data analysis were performed as described in section 2.5.1.
with the following changes:
⁻
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(a) Instead of a 1 mM Cl solution, samples were prepared using 100 µL of 1 mM solutions
3
2
containing the following ionic constituents: Cl
⁻
, I
⁻
, Br , F , PO4 , HCO3 , NO2 ,
⁻ ⁻ ⁻ ⁻ , CH COO⁻ , SO4 ⁻ ⁻
3
2
+
+
2+
2+
2+
2+
2+
NO3 , ClO4 , Ca , K , Mg , Cd , Cu , Fe , and Ni .
⁻ ⁻
1
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1
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(b) Samples were prepared using 100 µL of the following ionic constituents: [I
⁻
] = 1 µM, [Br
⁻
] = 1
] =
3
2
µM, [F
⁻
] = 1 µM, [PO4
⁻
] = 20 µM, [HCO3
⁻
] = 200 µM, [CH COO
⁻
] = 20 µM, [SO4
⁻
] = 20 µM, [NO2
⁻
3
2+
+
2+
2+
1 mM, [NO3
⁻
] = 1 mM, [ClO4
⁻
] = 1 mM, [Ca ] = 250 µM, [K ] = 500 µM, [Mg ] = 20 µM, [Cd ] = 1 µM,
2
+
2+
2+
[Cu ] = 1 µM, [Fe ] = 1 µM, and [Ni ] = 1 µM.
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2.5.4. Competing effect of ionic constituents
1
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To determine the competing effects of other ions on chloride ion binding, the experiments described in
⁻
sections 2.5.3. (a) and (b) were repeated with 1 mM Cl ion solution added to the samples.
1
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2.5.5. Quantitative determination of chloride ions in artificial sweat
1
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Sample preparation, measurements, and data analysis were performed as described in section 2.5.1.
using 10 µL artificial sweat solution containing different chloride ion concentrations (20 mM, 50 mM, 100 mM).
Artificial sweat solutions were prepared as previously described [51-53]. Quantitation was performed by
comparing the fluorescence recovery of the artificial sweat treated samples with the results obtained from the
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experiments in section 2.5.1. Chloride ion recovery was calculated by comparing the results with the estimated
concentrations obtained from the experiments in section 2.5.1.
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3. Results and discussion
3.1. Selection of optimal fluorescent chemosensor for chloride ions
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To identify the optimal Cl
⁻
ion fluorescent chemosensor, six different benzimidazole derivatives (1a-
c, 2a-c) with varying substituents at the C2 and C6 positions were synthesized and used as fluorescent ligands to
+
+
+
prepare Ag complexes. The fluorogenic response of the benzimidazole derivatives to Ag ions and the Ag -
benzimidazole complexes to chloride ions was then investigated (Figure 1). We first examined the change in
+
+
fluorescence intensity of the six benzimidazole derivatives upon the addition of Ag . In the absence of Ag , all
benzimidazole derivatives showed strong fluorescence peaks around 350 nm to 400 nm (Figure 1, Dye alone).
+
Following the addition of Ag , fluorescence intensity decreased, with saturation occurring for 2-5 equivalents of
+
+
Ag within 30 minutes (Figure 1, Ag -Dye w/o Cl
⁻
and Supporting Information). The quantum yields (Ф) of the
+
benzimidazole derivatives and the Ag -benzimidazole complexes were also determined (Table S1). To
+
investigate the recovery of fluorescence intensity of Ag -benzimidazole complexes in the presence of Cl
⁻
ions,
+
Cl
⁻
ions were added to solutions of the complexes, resulting in a notable increase in the fluorescence of Ag -1a
+
+
+
(~ 8 fold), Ag -2a (~ 11 fold), and Ag -2c (~ 7 fold) (Figure 1, Ag -Dye w/ Cl
⁻
). This indicated that the
+
fluorescence of the benzimidazole derivatives, which had been quenched upon complexation with Ag , was
+
recovered through ligand exchange between the Ag -dye complex and the chloride ions. This exchange can be
+
+
attributed to the strong affinity of Ag for Cl
⁻
ions forming AgCl. Based on these results, the Ag -2a complex
+
(Ag -FBI complex) was chosen as the optimal probe for the detection of Cl
⁻
ions.
7

1
98
+
1
2
2
2
99
00
01
02
Figure 1. Fluorescence intensity of benzimidazole derivatives (1a-1c, 2a-2c) complexed with Ag and in the
+
+
absence/presence of Cl
⁻
ions. Ag -Dye complex: red bar, [Dye]:[Ag ] = 25 µM:125 µM, 20 mM pH 8 HEPES,
+
+
10% DMSO. Ag -Dye complex with Cl
⁻
: blue bar, [Dye]:[Ag ]:[Cl
⁻
] = 25 µM:125 µM:1 mM, 20 mM pH 8
HEPES, 10% DMSO. Measurements taken at the specific λem of each benzimidazole derivative.
+
2
03
3.2. Ag -FBI complex as fluorescent probe for chloride ions
+
1
2
2
2
2
2
2
2
2
2
2
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06
07
08
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12
13
The chemical structure of the Ag -FBI complex was determined by FT-IR and H-NMR spectroscopy.
-1
The FT-IR spectra of free and complexed FBI showed a shift of the ν(C=N, benzimidazole) from 1420 cm to
-
1
+
1385 cm in the presence of Ag , indicating the involvement of the benzimidazole ring tertiary nitrogen in
1
coordination (Figure S23) [54,55]. A comparison between the H-NMR spectra of the free and complexed FBI
+
showed a downfield shift for the benzimidazole –NH from 12.95 ppm in the presence of Ag (Figure S24). In
addition, the aryl and furanyl moieties signal at 7.6-7.0 ppm also shifted and displayed a distinct splitting pattern
+
(Figure S24). These results suggest that the benzimidazole –NH remains intact during Ag complexation and
indicate that the furanyl ring is involved in the formation of the complex (Scheme 1, Fluorescence OFF structure)
+
[56,57]. This was further confirmed by a Job plot, which revealed a 1:1 binding stoichiometry between Ag and
FBI (Figure S25).
+
2
2
2
2
2
14
15
16
17
18
To determine the Cl
⁻
-sensing capacity of the Ag -FBI complex, the change in the fluorescence
intensity of the complex was measured in the presence of various chloride ion concentrations (Figure 2). Since
+
+
the decrease in the fluorescence intensity of FBI by Ag was saturated with 5 equivalents of Ag , 25 µM FBI
+
+
and 125 µM Ag were used to prepare the Ag -FBI complex in a pH 7.4 HEPES buffer solution containing 10%
+
DMSO (Figure S26). The fluorescence intensity of the Ag -FBI complex increased linearly with increasing
8

2
2
2
2
2
19
20
21
22
23
chloride ion concentration up to 1.5 mM (Figure 2b). The limit of detection (LOD) for chloride ions was
determined to be 19 µM based on the 3σ/slope (Figure 2b; σ 3.441), which is lower than for the majority of
previously reported Cl
⁻
probes. Most importantly, this detection limit is below the maximum concentration of
+
chloride ions in sweat, indicating that our Ag -FBI complex could be used for quantifying chloride ions in sweat
without interference from the biological matrix.
2
24
+
2
2
2
2
25
26
27
28
Figure 2. (a) Fluorescence spectra for incremental increases in chloride ion concentration ([FBI]:[Ag ] = 25
+
+
µM:125 µM). (b) Plot of the fluorescence intensity at 344 nm of Ag -FBI ([FBI]:[Ag ] = 25 µM:125 µM)
versus the concentration of Cl
⁻
(20 mM pH 7.4 HEPES, 10% DMSO, λex = 307 nm). F.I.: Fluorescence
intensity. LOD: Limit of detection.
+
2
2
2
2
2
2
2
2
2
29
30
31
32
33
34
35
36
37
Since sweat is composed of various ionic constituents, it is essential that the Ag -FBI complex
displays a high selectivity for chloride ions to be applicable for sweat-related applications. Both anions and
metal cations are constituents of sweat, and the effect of these ions on the detection of Cl
⁻
was examined. It
should be noted that, in physiological conditions, the concentrations of these other ions in sweat are much lower
than the level of chloride ion [15,51]. Though some of ion species at high concentrations were found to interfere
+
with the response of the Ag -FBI complex (Figure S27), in physiological conditions, other ions except for Cl
⁻
+
+
induced negligible change in fluorescence intensity of Ag -FBI complex. These results demonstrated that Ag -
+
FBI complex exhibits high selectivity toward Cl
⁻
, suggesting that Ag -FBI should be applicable for detecting
chloride ions in sweat, despite its contamination with other biological constituents.
9

2
38
+
+
2
2
2
2
2
2
39
40
41
42
43
44
Figure 3. Change in fluorescence intensity of Ag -FBI ([FBI]:[Ag ] = 25 µM:125 µM) in the presence of
3
biologically relevant ions, [Cl
[HCO3 ] = 200 µM, [CH COO
⁻
] = 1 mM, [I
⁻
] = 1 µM, [Br
⁻
] = 1 µM, [F
⁻
] = 1 µM, [PO4
⁻
] = 20 µM,
2
⁻
⁻
] = 1 mM, [SO4
⁻
] = 1 mM, [NO2
⁻
] = 1 mM, [NO3
⁻
] = 1 mM, [ClO4
⁻
] = 1
3
2
+
+
2+
2+
2+
2+
2+
mM, [Ca ] = 250 µM, [K ] = 500 µM, [Mg ] = 20 µM, [Cd ] = 1 µM, [Cu ] = 1 µM, [Fe ] = 1 µM, [Ni ] =
1 µM, (20 mM pH 7.4 HEPES, 10% DMSO, λ = 307 nm). F: fluorescence intensity. F : fluorescence intensity
ex
0
+
of Ag -FBI in the absence of ions.
2
2
2
2
45
46
47
48
The effect of pH on chloride ion detection was investigated in the pH range of 4.0-9.0 (Figure 4). It is
+
worth noting that while the Ag -FBI complex functioned well across a pH range of 6.0-8.0, the strongest
+
response to chloride ions was observed at the biologically relevant pH of 7.4. This finding suggests that the Ag -
FBI complex is applicable to chloride ion detection in physiological conditions.
1
0

2
49
+
2
2
2
2
50
51
52
53
Figure 4. Change of fluorescence intensity of FBI with silver ions (black bar; [FBI]:[Ag ] = 25 µM:125 µM).
+
Change of fluorescence intensity of Ag -FBI complex in the presence of chloride ions (red bar;
+
[FBI]:[Ag ]:[Cl
⁻
] = 25 µM:125 µM:1 mM). Conditions: 20 mM pH buffer, 10% DMSO, λex = 307 nm. F:
fluorescence intensity. F : fluorescence intensity of control.
0
2
54
3.3. Quantitative determination of chloride ions in artificial sweat
+
2
2
2
2
2
2
2
2
2
55
56
57
58
59
60
61
62
63
To evaluate the suitability of the Ag -FBI complex to detect chloride ions in sweat samples for the
diagnosis of CF, the chloride ion level of artificial sweat samples was determined (Table 1). Three artificial
sweat samples with different chloride ion concentrations were prepared following the British Standard (BS
EN1811-1999). Chloride ion concentrations were selected with respect to the diagnostic reference level for
cystic fibrosis in adults: < 40 mM (negative), 40–59 mM (possible), and > 60 mM (positive), with slightly lower
values for infants. Chloride ion recoveries ranged from 98 to 114%, with the relative standard deviation (RSD)
+
ranging between 4.55 to 7.22% (Table 1). These results indicate that the Ag -FBI complex accurately detects
chloride ions in complex artificial sweat samples, suggesting that this probe has great potential for use in the
sweat test diagnosis of cystic fibrosis.
⁻
2
64
Table 1. Detection and quantitation of Cl in artificial sweat samples.
-
a
[
Cl ]
Recovery (%)
RSD (%) (n=3)
Relative error (%)
2
0 mM
109 ± 3
4.55
2.63
11

5
0 mM
114 ± 5
98 ± 4
6.97
7.22
4.02
4.17
1
00 mM
a
2
2
65
66
Relative standard deviation.
4. Conclusions
+
+
2
2
2
2
2
2
2
2
2
67
68
69
70
71
72
73
74
75
We developed an Ag -benzimidazole complex-based chloride ion chemosensor, Ag -FBI, by exploiting the
+
⁻
+
strong interaction between Ag and Cl ions. The fluorescence intensity of the Ag -FBI complex increased in
⁻
the presence of Cl ions and allowed for the sensitive (LOD = 19 µM) and rapid (response time < 3 min)
⁻
+
detection of Cl ions. In addition, the Ag -FBI complex showed high selectivity toward Cl
⁻
ions and was
unaffected by other common ionic constituents in sweat. Notably, the chemosensor worked effectively in the pH
range of 6.0-8.0 with peak performance at the biologically relevant pH of 7.4 and chloride ions in complex
+
artificial sweat samples were quantitatively measured. The Ag -FBI complex is a simple and effective chloride
ion chemosensor and is expected to have clinical applications for quantifying chloride ions in diagnostic sweat
tests.
2
76
Acknowledgements
2
2
2
77
78
79
This work was supported by GIST Research Institute(GRI) grant funded by the GIST in 2019 and the National
Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (NRF-
2018R1A4A1024963).
2
2
2
80
81
82
Appendix A. Supplementary data
Supplementary data related to this article can be found at doi ****
References
2
2
83
84
[1] Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular Structure and Physiological Function of Chloride
Channels. Physiol Rev 2002;82:503–68. https://doi.org/10.1152/physrev.00029.2001
2
2
2
85
86
87
[2] Sweeney DW, Granade GV, Eversmeyer MG, Whitney DA. Phosphorous, Potassium, Chloride, and
Fungicide Effects on Wheat Yield and Leaf Rust Severity.
https://doi.org/10.1080/01904160009382099.
J
Plant Nutr 2000;23:1267–81.
2
88
[3] Iniesta J, González-García J, Expósito E, Montiel V, Aldaz A. Influence of chloride ion on electrochemical
1
2

2
2
89
90
degradation of phenol in alkaline medium using bismuth doped and pure PbO anodes. Wat Res 2001;35:3291–
2
300. https://doi.org/10.1016/S0043-1354(01)00043-4.
2
2
2
2
91
92
93
94
[4] Steffensen I-L, Frolich W, Dahl KH, Iversen PO, Lyche JL, Lillegaard ITL, Alexander J. Benefic and risk
assessment of increasing potassium intake by replacement of sodium chloride with potassium chloride in
industrial
food
products
in
Norway.
Food
Chem
Toxicol
2018;111:329–40.
https://doi.org/10.1016/j.fct.2017.11.044.
2
2
95
96
[5] LeGrys VA. Sweat testing for the diagnosis of cystic fibrosis: Practical considerations. J Pediatr
1996;129:892–7. https://doi.org/10.1016/s0022-3476(96)70034-3.
2
2
97
98
[6] LeGrys VA. Assessment of Sweat-Testing Practices for the Diagnosis of Cystic Fibrosis. Arch Pathol Lab
Med. 2001;125:1420–4. https://doi.org/10.1043/0003-9985(2001)125<1420:AOSTPF>2.0.CO;2.
2
3
99
00
[7] Puljak L, Kilic G. Emerging roles of chloride channels in human diseases. Biochim Biophys Acta-Mol Basis
Dis 2006;1762:404–13. https://doi.org/10.1016/j.bbadis.2005.12.008.
3
3
01
02
[8] LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel Jr PJ. Diagnostic Sweat Testing: The
Cystic Fibrosis Foundation Guidelines. J Pediatr 2007;151:85–9. https://doi.org/10.1016/j.jpeds.2007.03.002.
3
3
3
3
03
04
05
06
[9] Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N,
Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C. Identification of the Cystic Fibrosis Gene: Cloning
and
Characterization
of
Complementary
DNA.
Science
1989;245:1066–73.
https://doi.org/10.1126/science.2475911.
3
3
07
08
[10] Sheppard DN, Welsh MJ. Structure and Function of the CFTR Chloride Channel. Physiol Rev
1999;79:S23–45. https://doi.org/10.1152/physrev.1999.79.1.S23.
3
3
09
10
[11] López-Ruiz B. Advances in the determination of inorganic anions by ion chromatography. J Chromatogr A
2000;881:607–27. https://doi.org/10.1016/S0021-9673(00)00244-2.
3
3
3
11
12
13
[12] Shishkanova TV, Sýkora D, Sessler JL, Král V. Potentiometric response and mechanism of anionic
recognition of heterocalixarene-based ion selective electrodes. Anal Chim Acta 2007;587:247–53.
https://doi.org/10.1016/j.aca.2007.01.044.
3
14
[13] Kimmel DW, LeBlanc G, Meschievitz ME, Cliffel DE. Electrochemical Sensors and Biosensors. Anal
1
3

3
15
Chem 2012;84:685–707. https://doi.org/10.1021/ac202878q.
3
3
3
16
17
18
[14] Toh HS, Batchelor-McAuley C, Tschulik K, Compton RG. Electrochemical detection of chloride levels in
sweat using silver nanoparticles: a basis for the preliminary screening for cystic fibrosis. Analyst
2013;138:4292–7. https://doi.org/10.1039/c3an00843f.
3
3
3
19
20
21
[15] Lopez-Moreno C, Perez IV, Urbano AM. Development and validation of an ionic chromatography method
for the determination of nitrate, nitrite and chloride in meat. Food Chem 2016;194:687–94.
https://doi.org/10.1016/j.foodchem.2015.08.017.
3
3
22
23
[16] Cunha-Silva H, Acros-Martinez J. Development of a selective chloride sensing platform using a screen-
printed platinum electrode. Talanta 2019;195:771–7. https://doi.org/10.1016/j.talanta.2018.12.008.
3
3
24
25
[17] Bai Y, Zhang B-G, Xu J, Duan C-Y, Dang D-B, Liu D-J, Meng Q-J. Conformational switching fluorescent
chemosensor for chloride anion. New J Chem 2005;29:777–9. https://doi.org/10.1039/B500252D.
3
3
3
26
27
28
[18] Schazmann B, Alhashimy N, Diamond D. Chloride Selective Calix[4]arene Optical Sensor Combining
Urea Functionality with Pyrene Excimer Transduction.
https://doi.org/10.1021/ja061917m.
J
Am Chem Soc 2006;128:8607–14.
3
3
3
29
30
31
[19] Wu J-S, Wang P-F, Zhang X-H, Wu S-K. An efficient chloride-selective fluorescent chemosensor based on
2,9-bis(4’-hydroxyphenyl)phenanthroline Cu(II) complex. Spectroc Acta Pt
https://doi.org/10.1016/j.saa.2006.07.021.
A
2007;67:281–6.
3
3
3
32
33
34
[20] Tavallali H, Rad GD, Parhami A, Abbasiyan E. Colorimetric detection of copper and chloride in
DMSO/H O media using bromopyrogallol red as a chemosensor with analytical applications. Spectroc Acta Pt
2
A-Molec Biomolec Spectr 2012;97:60–5. https://doi.org/10.1016/j.saa.2012.05.071.
3
3
3
35
36
37
[21] Kaur A, Sharma H, Kaur S, Singh N, Kaur N. A counterion displacement assay with a Biginelli product: a
2
+
-
ratiometric sensor for Hg and the resultant complex as a sensor for Cl . RSC Advances 2013;3:6160–6.
https://doi.org/10.1039/c3ra40236c.
3
3
38
39
[22] Dorazco-Gonzalez A. Chemosensing of Chloride Based on a Luminescent Platinum(II) NCN Pincer
Complex in Aqueous Media. Organometallics 2014;33:868–75. https://doi.org/10.1021/om4007054.
3
40
[23] Bazany-Rodríguez I, Martínez-Otero D, Barroso-Flores J, Yatsimirsky AK, Dorazco-González A. Sensitive
1
4

3
3
41
42
water-soluble fluorescent chemosensor for chloride based on a bisquinolinium pyridine-dicarboxamide
compound. Sens Actuator B-Chem 2015;221:1348–55. https://doi.org/10.1016/j.snb.2015.07.031.
3
3
43
44
[24] Chilbac AL, Roman G, Cojocaru C, Sacarescu G, Simionescu M, Sacarescu L. Pyrazoline based chloride
sensor for body fluids screening. J Mol Liq 2019;284:139–46. https://doi.org/10.1016/j.molliq.2019.04.007.
2
+
-
3
3
3
45
46
47
[25] Tong L, Sun S, Qiao M, Fan J, Ding L. Selective turn-on sensing of Cu and Cl by a ferrocene-modified
pyrene derivative. Photochem Photobiol A-Chem 2019;374:131-7.
https://doi.org/10.1016/j.jphotochem.2019.01.036.
J
3
3
3
48
49
50
[26] Li P, Xie T, Fan N, Li K, Tang B. Ratiometric fluorescence imaging for distinguishing chloride
concentration between normal and ischemic ventricular myocytes. Chem Commun 2012;48:2077–9.
https://doi.org/10.1039/C1CC15258K.
3
3
51
52
[27] Kim JP, Xie Z, Creer M, Liu Z, Yang J. Citrate-based fluorescent materials for low-cost chloride sensing in
the diagnosis of cystic fibrosis. Chem Sci 2017;8:550–8. https://doi.org/10.1039/c6sc02962k.
3
3
53
54
[28] Zhang F, Ma C, Wang Y, Liu W, Liu X, Zhang H. Fluorescent probes for chloride ions in biological samples.
Spectroc Acta Pt A-Molec Biomolec Spectr 2018;205:428–34. https://doi.org/10.1016/j.saa.2018.07.060.
3
3
3
55
56
57
[29] Vallejos S, Hernando E, Trigo M, Garcia FC, Garcia-Valverde M, Iturbe D, Cabero MJ, Quesada R, Garcia
JM. Polymeric chemosensor for the detection and quantification of chloride in human sweat. Application to the
diagnosis of cystic fibrosis. J Mater Chem B 2018;6:3735–41. https://doi.org/10.1039/c8tb00682b.
3
3
58
59
[30] Ma C, Zhang F, Wang Y, Zhu X, Liu X, Zhao C, Zhang H. Synthesis and application of ratio fluorescence
probe for chloride. Anal Bioanal Chem 2018;410:6507–16. https://doi.org/10.1007/s00216-018-1250-0.
3
3
60
61
[31] Jonte JH, Martin Jr DS. The Solubility of Silver Chloride and the Formation of Complexes in Chloride
Solution. J Am Chem Soc 1952;74:2052–4.
3
62
[32] Harris DC. Quantitative Chemical Analysis. 8th ed. W.H. Freeman Custom Publishing; 2010.
3
3
63
64
[33] Meija J. Mohr’s method challenge. Anal Bioanal Chem 2016;408:1721–2. https://doi.org/10.1007/s00216-
015-9273-2.
3
3
65
66
[34] Dean KES, Klein G, Renaudet O, Reymond J-L. A Green Fluorescent Chemosensor for Amino Acids
Provides a Versatile High-Throughput Screening (HTS) Assay for Protease. Bioorg Med Chem Lett
1
5

3
67
2003;13:1653-6. https://doi.org/10.1016/S0960-894X(03)00280-4.
3
3
68
69
[35] Chen Y, Chen J, Ma K, Cao S, Chen X. Fluorimetric determination of phytic acid in urine based on
replacement reaction. Anal Chim Acta 2007;605:185–91. https://doi.org/10.1016/j.aca.2007.10.041.
3
3
3
70
71
72
[36] Lou X, Zhang L, Qin J, Li Z. An alternative approach to develop a highly sensitive and selective
chemosensor for the colorimetric sensing of cyanide in water. Chem Commun 2008;5848–50.
https://doi.org/10.1039/B812746H.
2
+
3
3
73
74
[37] Choi MG, Cha S, Lee H, Jeon HL, Chang S-K. Sulfide-selective chemosignaling by a Cu complex of
dipicolylamine appended fluorescein. Chem Commun 2009;7390–2. https://doi.org/10.1039/B916476F.
3
3
75
76
[38] Jung HS, Han JH, Habata Y, Kang C, Kim JS. An iminocoumarin-Cu(II) ensemble-based chemodosimeter
toward thiols. Chem Commun 2011;47:5142-4. https://doi.org/10.1039/c1cc10672d.
3
3
3
77
78
79
[39] Kittiloespaisan E, Takashima I, Kiatpathomchai W, Wongkongkatep J, Ojida A. Coordination ligand
exchange of a xanthene probe-Ce(III) complex for selective fluorescence sensing of inorganic pyrophosphate.
Chem Commun 2014;50:2126–8. https://doi.org/10.1039/c3cc48101h.
3
3
3
80
81
82
[40] Eom MS, Noh J, Kim H-S, Yoo S, Han MS, Lee S. High-Throughput Screening Protocol for the Coupling
Reactions of Aryl Halides Using a Colorimetric Chemosensor for Halide Ions. Org Lett 2016;18:1720-3.
https://doi.org/10.1021/acs.orglett.6b00300.
3
3
3
83
84
85
[41] Lee S, Kang S, Eom MS, Han MS. Colorimetric assay for β-lactamase activity using cocktail of penicillin
2
+
and
4-(2-pyridylazo)resorcinol
(PAR)-2Hg
complex.
Dyes
Pigment
2017;137:518-22.
https://doi.org/10.1016/j.dyepig.2016.10.026.
3
3
3
86
87
88
[42] McCann M, Curran R, Ben-Shoshan M, McKee V, Devereux M, Kavanagh K, Kellett A. Synthesis,
structure and biological activity of silver(I) complexes of substituted imidazoles. Polyhedron 2013;56:180–8.
https://doi.org/10.1016/j.poly.2013.03.057.
3
3
3
89
90
91
[43] Wang XX, Liu YG, Hecke KV, Goltsev A, Cui GH. Three Silver(I) Coordinatino Polymers Constructed
from Flexible Bis(benzimidazole) and Carboxylates Ligands. Z Anorg Allg Chem 2015;641:903–10.
https://doi.org/10.1002/zaac.201400597.
3
92
[44] Haque RA, Choo SY, Budagumpi S, Iqbal MA, Abdullah AA. Silver(I) complexes of mono- and bidentate
1
6

3
3
93
94
N-heterocyclic carbine ligands: Synthesis, crystal structures, and in vitro antibacterial and anticancer studies.
Eur J Med Chem 2015;90:82–92. https://doi.org/10.1016/j.ejmech.2014.11.005.
3
3
3
3
95
96
97
98
[45] Kalinowska-Lis U, Felczak A, Chęcińska L, Szabłowska-Gadomska I, Patyna E, Małecki M, Lisowska K,
Ochocki J. Antibacterial Activity and Cytotoxicity of Silver(I) Complexes of Pyridine and (Benz)Imidazole
Derivatives. X-ray Crystal Structure of [Ag(2,6-di(CH OH)py) ]NO . Molecules 2016;21:87–100.
2
2
3
https://doi.org/10.3390/molecules21020087.
3
4
4
99
00
01
[46] Liang X, Luan S, Yin Z, He M, He C, Yin L, Zou Y, Yuan Z, Lixia Y, Song X, Lv C, Zhang W. Recent
advances in the medical use of silver complex. Eur
https://doi.org/10.1016/j.ejmech.2018.07.057.
J
Med Chem 2018;157:62–80.
4
4
4
02
03
04
[47] Jiang Y-Q, Jia S-H, Sun Y-M, Li Wei, Zhang W-W, Xu G-Q. An efficient NaHSO -promoted protocol for
3
chemoselective synthesis of 2-substituted benzimidazoles in water. Chem Pep 2018;72:1265–76.
https://doi.org/10.1007/s11696-017-0367-5.
4
4
4
05
06
07
[48] Xiang S-K, Tan W, Zhang D-X, Tian X-L, Feng C, Wang B-Q, Zhao K-Q, Hu P, Yang H. Synthesis of
benzimidazoles by potassium tert-butoxide-promoted intermolecular cyclization reaction of 2-iodoanilines with
nitriles. Org Biomol Chem 2013;11:7271–5. https://doi.org/10.1039/c3ob41479e.
4
08
[49] Valeur B. Molecular Fluorescence: Principles and Applications. Wiley-VCH Verlag GmbH; 2001.
4
4
4
09
10
11
[50] Huang S, He S, Lu Y, Wei F, Zeng X, Zhao L. Highly sensitive and selective fluorescent chemosensor for
+
Ag
based on
a
coumarin-Se N chelating conjugate. Chem Commun 2011;47:2408–10.
2
https://doi.org/10.1039/C0CC04589F.
4
4
12
13
[51] Stefaniak AB, Harvey CJ. Dissolution of materials in artificial skin surface film liquids. Toxicol Vitro
2006;20:1265–83. https://doi.org/10.1016/j.tiv.2006.05.011.
4
4
4
14
15
16
[52] Kulthong K, Srisung S, Boonpavanitchakul K, Kangwansupamonkon W, Maniratanachote R.
Determination of silver nanoparticle release from antibacterial fabrics into artificial sweat. Part Fibre Toxicol
2010;7:8–16. https://doi.org/10.1186/1743-8977-7-8.
4
4
17
18
[53] Callewaert C, Buysschaert B, Vossen E, Fievez V, Van de Wiele T, Boon N. Artificial sweat composition to
grow and sustain
a
mixed human axillary microbiome.
J
Microbiol Methods 2014;103:6–8.
1
7

4
19
https://doi.org/10.1016/j.mimet.2014.05.005.
4
4
4
20
21
22
[54] Galal SA, Hegab KH, Hashem AM, Youssef NS. Synthesis and antitumor activity of novel benzimidazole-
5-carboxylic acid derivatives and their transition metal complexes as topoisomerase II inhibitors. Eur J Med
Chem 2010;45:5685-91. https://doi.org/10.1016/j.ejmech.2010.09.023.
4
4
4
23
24
25
[55] Haque RA, Iqbal MA, Khadeer Ahamed MB, Majid AA, Hameed ZAA. Design, synthesis and structural
studies of meta-xylyl linked bis-benzimidazolium salts: potential anticancer agents against ‘human colon
cancer’. Chem Cent J 2012;6:68-81. https://doi.org/10.1186/1752-153X-6-68.
4
4
4
26
27
28
[56] Fisher CM, Fuller E, Burke BP, Mogilireddy V, Pope SJA, Sparke AE, Déchamps-Olivier I, Cadiou C,
Chuburu F, Faulkner S, Archibald SJ. A benzimidazole functionalized DO3A chelator showing pH switchable
coordination modes with lanthanide ions. Dalton Trans 2014;43:9567–78. https://doi.org/10.1039/c4dt00519h.
4
4
4
4
29
30
31
32
[57] Majumdar A, Mondal S, Daniliuc CG, Sahu D, Ganguly B, Ghosh S, Ghosh U, Ghosh K. α-Amino Acid
Derived Benzimidazole-Linked Rhodamines: A Case of Substitution Effect at the Amino Acid Site toward Spiro
3+
Ring
Opening
for
Selective
Sensing
of
Al
Ions.
Inorg
Chem
2017;56:8889−99.
https://doi.org/10.1021/acs.inorgchem.7b00835.
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Highlights
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•
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Ag –benzimidazole (FBI) complex was developed as fluorescent chemosensor
for Cl⁻ .
The probe exhibited high selectivity and sensitivity toward Cl⁻ (detection
limit 19 µM).
The probe was successfully applied to the detection of Cl⁻ in artificial sweat.

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.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: