Novel styrylbenzimidazolium-based fluorescent probe for reactive sulfur species: Selectively distinguishing between bisulfite and thiol amino acids

Article abstract of DOI:10.1016/j.saa.2021.120151

In this study, a new fluorescent probe containing dicyanovinyl moiety has been designed and synthesized. Fluorescent probe based on styrylbenzimidazolium derivative was reported for the effective detection of bisulfite and distinguish it from biothiols by exploiting dicyanovinyl group as the recognition site. The photophysical properties of the novel styrylbenzimidazolium derivative were assessed by determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. Its spectroscopic behavior towards various analytes has been evaluated in aqueous media at a pH of 7.4. The highest increase in fluorescence intensity of compound 5 in the presence of different analytes was observed for sodium bisulfite and the limit of detection was estimated to be 0.25 μM. The styrylbenzimidazolium dye was applied to detect bisulfite in various wine sample using fluorimetry. Finally, the ability of this novel probe to detect HSO₃^? in red wine samples was evaluated.

Full text of DOI:10.1016/j.saa.2021.120151

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel styrylbenzimidazolium-based fluorescent probe for reactive sulfur
species: Selectively distinguishing between bisulfite and thiol amino
acids
⇑
⇑
´
Jolanta Kolinska , Aleksandra Grzelakowska
Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, 90-924 Lodz, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
New styrylbenzimidazolium
derivative with dicyanovinyl moiety
exhibit sensitivity and selectivity for
HSO3ˉ and discriminative
fluorescence sensing of Cys/Hcy over
GSH.
ꢀ
ꢀ
The detection limits of HSO3ˉ was
estimated to be 0.25 mM.
The styrylbenzimidazolium
derivative was used to detect bisulfite
in real samples.
ꢀ
The cytotoxicity of
styrylbenzimidazolium derivative
was examined against the HeLa cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2021
Received in revised form 30 June 2021
Accepted 1 July 2021
Available online 6 July 2021
In this study, a new fluorescent probe containing dicyanovinyl moiety has been designed and synthe-
sized. Fluorescent probe based on styrylbenzimidazolium derivative was reported for the effective detec-
tion of bisulfite and distinguish it from biothiols by exploiting dicyanovinyl group as the recognition site.
The photophysical properties of the novel styrylbenzimidazolium derivative were assessed by determi-
nation of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime.
Its spectroscopic behavior towards various analytes has been evaluated in aqueous media at a pH of
7.4. The highest increase in fluorescence intensity of compound 5 in the presence of different analytes
Keywords:
Fluorescent probe
Sodium bisulfite
Styrylbenzimidazolium
was observed for sodium bisulfite and the limit of detection was estimated to be 0.25 lM. The styrylben-
zimidazolium dye was applied to detect bisulfite in various wine sample using fluorimetry. Finally, the
ability of this novel probe to detect HSOꢁ3 in red wine samples was evaluated.
Ó 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
mented products and fruit/vegetable preserves. For several reason
sulfites are added during vinification. They eliminate harmful
Sulfites are naturally present in many foods, such as eggs, some
fish, asparagus, lettuce, garlic, onions, soybeans, and tomatoes.
Also, they are very often found in sugar, wine, and generally in fer-
microorganisms and wild yeasts that can cause incorrect fermenta-
tion. Sulfites also prevent wine from oxidation because they are
stabilizers and have preservative properties [1,2]. Moreover, in
white wine, they act as bleaching agents that maintain the desired
color of the wine [3]. Simultaneously, sulfites are popular food
products additives. They are also components of acid rain or smog
inhaled by residents of large urban agglomerations [4]. Except that,
⇑
Corresponding authors.
E-mail addresses: jolanta.kolinska@p.lodz.pl (J. Kolin´ ska), aleksandra.grzela-
kowska@p.lodz.pl (A. Grzelakowska).
https://doi.org/10.1016/j.saa.2021.120151
1386-1425/Ó 2021 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

´
J. Kolinska and A. Grzelakowska
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
sulfites are also used as the additives of some pharmaceuticals and
cosmetics during production and storage [5]. Due to the wide-
spread using of sulfates, World Health Organization has estab-
lished a safe daily limit of sulfur dioxide consumption at 0.7 mg
per kilogram of body weight [6]. The certain concentration level
of sulfur compounds are especially dangerous for asthmatics
because they can cause very strong allergic reactions, e.g. breathing
problems. According to the epidemiological research, an excess
sulfur dioxide in consumed products can reduce blood pressure
and cause bronchitis [7]. The sulfur compounds also destroy the
vitamins providing with food, especially A, B1 and B12. Undoubt-
edly, sulfites are present in many areas of life, so it is important
to detect and control their quantity. Hence, many different analyt-
ical methods have been used to determine sulfur derivatives
including electrochemistry, conductivity, chromatography, flow
injection analysis [1,8–12]. Fluorescent probes have become very
useful and efficient tools for the detection and the identification
of various analytes in food and beverages. In the last five years,
many fluorescent probes have been reported for the detection of
bisulfite [13]. They are mainly fluorescent probes based on through
different chemical reaction mechanisms, such as Michael addition
[14–16], nucleophilic reaction to aldehyde moiety [17–19], and
other reaction [20–22]. All of these mechanisms exploit the strong
nucleophilicity and reducing properties of sulfites to achieve selec-
tivity over other reactive sulfur species. The presence of strongly
electron-withdrawing group such as dicyanovinyl group in the flu-
orescent probe structure reduces the electron density of the car-
bon–carbon double bond and, therefore, it may undergo
nucleophilic attack. Various types of nucleophiles, including thiol
compounds [23–25], reactive sulfur species [26–30], and cyanide
anions [31,32] react with this kind of compounds.
of-flight mass analyser (Synapt G2-Si mass spectrometer, Waters).
UV–vis absorption spectra and fluorescence spectra were recorded
using a Jasco V-670 UV–vis/NIR spectrophotometer (Jasco, Japan)
and a FLS-920 spectrofluorometer (Edinburgh Instruments, UK),
respectively. In each case quartz cuvettes (1 cm) were used. The
excitation wavelength for fluorescence measurements was
395 nm (slit width: ex/em = 1.5 nm). The pH values were deter-
mined with a CPI-551 microcomputer pH/ion meter (Elmetron,
Poland)
2.2. Preparation of compound 5
2.2.1. Synthesis of trans-2-(4-formylstyryl)-[1H]-benzimidazole (3)
[34,35]
A mixture of 2-methyl-[1H]-benzimidazole 1 (1.32 g, 0.01 mol),
terephthalaldehyde 2 (1.34 g, 0.01 mol), acetic acid (1.5 ml), and
acetic anhydride (3 ml) was stirred at 120 °C for 20 h. The reaction
progress was monitored by TLC (eluent: toluene/ethanol 3:1 (v/v),
Rf = 0.73). Then, the solution was cooled to room temperature and
30% hydrochloric acid (15 ml) was added and after that the solu-
tion was filtered. The filtrate was neutralized with 30% sodium
hydroxide solution (30 ml) and resulted a yellowish precipitate
that was filtered and dried. The crude product 3 was recrystallized
from ethanol to yield 1.10 g (44%); mp 178–180 °C.
2.2.2. Synthesis of 2-(4-dicyanovinylstyryl)-[1H]-benzimidazole (5)
Malononitrile 4 (0.03 g, 0.5 mmol) was added to a solution of
trans-2-(4-formylstyryl)-[1H]-benzimidazole 3 (0.124 g, 0.5 mmol)
in absolute ethanol (15 ml), and then three drops of piperidine
were added to the mixture. The reaction mixture was stirred under
reflux until the disappearance of starting compounds (15 h), which
was checked by TLC using toluene/ethanol (3:1, v/v) as eluent
(Rf = 0.58). Then the solution was cooled to room temperature
and the resulting precipitate was filtered and dried. The crude pro-
duct 5 was recrystallized from ethanol to give an orange solid
(0.04 g, 28%); mp greater than 360 °C without decomposition.
1H NMR (DMSO d6, 250 MHz) d 7.24–7.17 (m, 2H), 7.64–7.50
(m, 2H), 7.47 (d, J = 16.5 Hz, 1H), 7.78 (d, J = 16.5 Hz, 1H), 7.91
(d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.5 Hz, 2H), 8.53 (s, 1H), 13.00
(brs, 1H).
Recently, we have synthesized and characterized in detail
styrylbenzimidazolium dye containing a maleimide moiety [33].
Here, we report the synthesis and characterization of a novel
styrylbenzimidazolium-based derivative with dicyanovinyl group
at the site of the maleimide moiety.
Then, we characterized and validated this compound as probe
for the direct detection of bisulfite over other sulfur species. Com-
pared with the previously reported probe for compounds contain-
ing a sulfhydryl group, probe 5 is able to distinguish different
reactive sulfur species.
13C NMR (DMSO d6, 62 MHz) d 80.7, 87.4, 113.6, 114.5, 121.3,
121.6, 122.5, 127.9, 130.2, 131.2, 131.4, 132.8, 135.4, 141.7,
150.4, 160.5. HRMS (MS ESI) m/z: [M
19H12N4 297.1140, found 297.1141.
+
H]+ calcd. for
2. Materials and methods
C
2.1. General
2.3. Fluorescence quantum yield and fluorescence lifetime
All chemicals were purchased from commercial suppliers
(Sigma-Aldrich, Poland) and used directly without further refine-
ment. Melting points were determined on the Boeöthius melting
point apparatus and uncorrected. The reaction progress and the
purity of the obtained compounds were routinely checked by thin
layer chromatography (TLC) on silica gel 60 F254 plates (Merck,
Germany). Plates were visualized with UV light (k = 254 nm or
365 nm). Crude materials were purified by column chromatogra-
phy on silica gel 60 (0.063–0.200 mm, Merck, Germany). 1H NMR
and 13C NMR spectra were obtained with tetramethylsilane
(TMS) as the internal standard and dimethylsulfoxide (DMSO d6)
as the solvent on a Bruker Avance DPX 250 (Germany) spectrome-
ter. The chemical shifts (d values) and coupling constants (J values)
are expressed in parts per million (ppm) and in hertz (Hz), respec-
tively. NMR peak multiplicities are described as follows: s (singlet),
d (doublet), m (multiplet), brs (broad singlet). High-resolution
mass spectrometry (HRMS) experiments were performed with a
mass spectrometer equipped with an electrospray ionization
source operated in the positive ion mode and quadrupole-time-
For the determination of fluorescence quantum yields, fluores-
cein in 0.1 M NaOH (Ust = 0.85) [36] was used as the standard. Flu-
orescence quantum yields
(
U
)
of tested compound
5
were
determined using following Eq. (1):
ꢀ
ꢁ
ꢀ
ꢁ
Gradsensor
Gradst
g2sensor
g2st
/ ¼ /st
ꢂ
ð1Þ
where the subscripts ‘st’ and ‘sensor’ denote the standard fluo-
rescein and the tested compound, respectively. is the quantum
U
yield of fluorescence, Grad is the gradient obtained from the plot
of the integrated fluorescence intensity as a function of the absor-
bance, at their excitation wavelengths, and
tive index.
g is the solvent refrac-
Fluorescence lifetime measurements were made using a time–
correlated single photon counting system (TCSPC) with a pulsed
picosecond diode (EPLED-380) as the excitation source. The instru-
ment response function was measured by collecting scattered light
from a Ludox silica suspension. Fluorescence lifetimes were calcu-
2

´
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
J. Kolinska and A. Grzelakowska
lated from intensity decay analyses. Fluorescence decay was
acquired at a level of 1 ꢂ 104 counts at the peak and was fitted
to a sum of exponentials (2):
and treated with the indicated concentrations (0, 6.25, 12.5,
18.75
lm) of compound 5 for 24 h. Cells viability was quantified
with PrestoBlue reagent from Life Technologies (Van Allen Way,
CA, USA) according to the procedure described in literature [38].
The cytotoxicity of the compound 5 was determined by measuring
the fluorescent signal at kex = 530 nm and kem = 590 nm using a
Synergy 2 Microplate Reader (Bio-Rad, CA, USA). The obtained flu-
orescence values were used to calculate cell viability expressed as
the percentage of viability of the untreated control.
n
X
IðtÞ ¼
a
iexpðꢁt=si
Þ
ð2Þ
i. Average lifetimes (
i¼1
with amplitudes,
a
i, and decay lifetimes
s
s)
for multiexponential fluorescence decay were calculated from
decay times and pre-exponential factors using (3):
P
ais2i
aisi
2.7. Preparation of wine samples
P
ð
sÞ ¼
ð3Þ
The three types of wine were selected: rose wine 1 (semi-dry
type), red wine 2 (semi-dry type), red wine 3 (sweet type) for
the practical study. The wine sample (1 ml) was mixed (1:100)
with distilled water. Then 0.3 ml of the prepared solution was
added to the solution of compound of 5 in phosphate buffer (10%
CH3CN, pH 7.4). For the control experiment, 10 mM of HSOꢁ3 was
added to each test solution and, the fluorescence intensity of all
wine samples at 410 nm were recorded.
2.4. General procedure for preparation of probe test solutions
L-cysteine (L-Cys), L-homocysteine (L-Hcy), L-glutathione (L-
GSH), N-acetyl-L-cysteine (L-ACC), Glycine (Gly), L-glutamic acid
(L-Glu), L-lysine (L-Lys), 2-mercaptoethanol (ME), thioglycolic acid
(TGA), sodium bisulfite (NaHSO3), sodium hydrosulfide (NaSH),
sodium sulfide (Na2S), hydrogen peroxide (H2O2), and N-
ethylmaleimide (NEM, a trapping reagent for thiol species [37]
stock solutions were prepared in distilled water (30 mM). The
stock solutions were diluted with distilled water to the desired
concentration and used immediately after preparation. For all
experiments a stock solution of 5 was prepared by dissolving accu-
rately weighted the solid compound in acetonitrile (CH3CN, 1 mM
5). The stock solution of probe was diluted with CH3CN to acquired
0.1 mM dye solution. All the detection experiments were per-
formed in phosphate buffer (0.1 M, pH 7.4) at room temperature.
Because of the relatively poor solubility of the 5 probe in water,
CH3CN was used as a cosolvent (10%; v/v). Additionally, in the
pH studies, an acetate buffer (0.1 M, pH 3.6–5.6), and carbonate-
bicarbonate buffer (0.1 M, pH 9.5–10.5) were also used.
In a typical experiment, the concentration of the probe 5 was
10 mM. The procedure was as follows: 0.3 ml of appropriate dye
solution (5) was added to 2.4 ml phosphate buffer (0.1 M, pH
7.4) and 0.3 ml of analyte solution was added. For the control
experiments, a solution of L-Cys (30 mM was pretreated with a
solution of NEM (30 mM) and incubated for 2 h at ambient temper-
ature (20–23 °C), and then the reaction mixture was added to the
test solution containing compound 5. The probe was incubated
with an appropriate amount of each analyte in a phosphate buffer
(0.1 M, pH 7.4) for 5 min. Then, absorption and fluorescence spec-
tra were recorded.
3. Results and discussion
3.1. Synthesis and spectroscopic characterization of 5
2-(4-dicyanovinylstyryl)-[1H]-benzimidazole 5 was prepared
by a simple two-step synthetic route as shown in Scheme 1. In
the first stage, the reaction between 2-methyl-[1H]-
benzimidazole 1 with terephthalaldehyde 2 was carried out by
refluxing in acetic anhydride – acetic acid mixture [34,35]. The
compound 3 was obtained in good yield (44%). Finally, compound
5 was synthesized by condensation of trans-2-(4-formylstyryl)-
[1H]-benzimidazole 3 with malononitrile 4 in absolute ethanol in
the presence of catalytic amounts of piperidine. The crude com-
pound 5 was purified by column chromatography and recrystal-
lization from ethanol until a TLC purity was obtained. 1H NMR,
13C NMR spectroscopy and ESI MS spectrometry studies evidenced
the chemical structure of probe 5 (Fig. S1).
The analysis the chemical shifts, multiplicities, and integration
of the relevant groups of protons are in accordance with the struc-
ture of the molecule. As expected, in the structure of 5 a styryl scaf-
fold have a trans configuration, as evidenced by the value of
coupling constants for vinyl protons (J = 16.5 Hz). In addition, the
characteristic broad singlet about 13 ppm derived from the proton
of NH group is observed.
The spectroscopic properties of compound 5 in pure acetonitrile
and in aqueous solution (10% CH3CN, pH 7.4) were studied by
means UV–vis absorption and fluorescence spectroscopy. The data
obtained are summarized in Table 1 and compared with styrylben-
zimidazolium derivative possessing maleimide group [33]. The
normalized absorption and fluorescence spectra of compound 5
were provided in Fig. S2.
Dye 5 exhibits an absorption band with a maximum at 390 nm
at pH 7.4. The electron-withdrawing group in the structure of com-
pound 5 affect the absorption and fluorescence properties. The
replacement of a maleimide group by dicyanovinyl moiety cause
a bathochromic shift of kabs and kem. The value of Stokes shift of
compound 5 is greater than 100 nm suggest that the geometries
of two states (ground and excited) are different. It should be
All experiments were carried out at least in triplicate and the
results are presented as means standard deviation.
2.5. Determination of the detection limit
The limit of detection for styrylbenzimidazolium derivative was
calculated according to the definition by IUPAC (3r/m, where r is
the standard deviation of the blank measurement of compound 5
and m is the slope of a plot of fluorescence intensity against HSO3ꢁ
concentration). In the absence of analytes, the fluorescence emis-
sion spectrum of probe 5 was measured five times and the stan-
dard deviation of blank measurement was achieved.
2.6. Cell viability
The human cell line HeLa was obtained from CBMM PAN Lodz
and was cultured in RPMI (Roswell Park Memorial Institute
1640) medium with 10% fetal bovine serum (FBS, Invitrogen) sup-
plemented with penicillin and streptomycin antibiotic at 37 °C in
5% CO2 air atmosphere/95% air atmosphere. The cells (1 ꢂ 104 cells
emphasized that probes with a large Stokes shift (greater
than80 nm) are essential for biological applications due to minimal
self-quenching and scattered light during fluorescence imaging.
Moreover, the compound with dicyanovinyl group possess higher
fluorescence quantum yield (
U) and longer lifetime of singlet
per 100
l
L) were plated into each well of 96-well microtitre plates
excited state ( ) compared with previously described probe with
s
3

´
J. Kolinska and A. Grzelakowska
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
Scheme 1. The synthesis of compound 5.
Table 1
Spectroscopic characterization of compounds with dicyanovinyl and maleimide group in CH3CN(a) and in phosphate buffer (0.1 M, pH 7.4) containing 10% CH3CN(b)
.
kabs [nm]
e
[Mꢁ1*cmꢁ1
]
kem [nm]
U
s
[ns]
SS [nm]
395(a)
390(b)
14300(a)
13300(b)
601(a)
550(b)
0.58(a)
0.05(b)
2.27(a)
0.55(b)
206(a)
160(b)
N
NC
CN
NH
N
339(a)
339(b)
25300(a)
24400(b)
409(a)
410(b)
0.20(a)
0.07(b)
1.36(a)
-
70(a)
71(b)
O
O
NH
N
340(b)
14100(b)
410(b)
0.44(b)
1.29(b)
70(b)
N
NC
CN
H
NH
SO
3
maleimide moiety. The fluorescence decay profile of
described by single-exponential fit (Fig. S3).
5
was
changes to the UV–vis absorption spectrum. The visible absorption
band located ca. 400 nm was gradually disappeared and the
appearance of a new peak ca. 346 nm, with an isosbestic point at
365 nm, which indicates the formation of a new reaction product
between 5 and bisulfite, was observed. The hipsochromic effect
in the fluorescence emission spectra after the addition of bisulfite
to the solution of compound 5 was also observed. More impor-
tantly, the fluorescence intensity of the product formed in the reac-
tion between the probe 5 and bisulfite at 410 nm is over 68-fold
higher in comparison with the fluorescence intensity of 5 at the
same wavelength.
3.2. Studies of the fluorescent response to bisulfite
Compounds possessing dicyanovinyl moiety are susceptible to
nucleophilic attack by several nucleophiles [23–32]. As is well
known, the presence of dicyanovinyl moiety in the molecule is
used as recognition of bisulfite [26–29]. Thus, UV–vis absorption
spectra and fluorescence emission spectra of compound 5 upon
the addition of NaHSO3 in phosphate buffer (10% CH3CN, pH 7.4)
were measured and are shown in Fig. 1. It was observed that the
addition of bisulfite to solutions of compound 5 causes significant
In order to determine the limit of detection of compound 5
against HSOꢁ3 in phosphate buffer (10% CH3CN, pH 7.4), the absorp-
Fig. 1. (A) Absorption (B) fluorescence spectra changes of 5 (10 mM) in phosphate buffer (10% CH3CN, pH 7.4) after addition of bisulfite (3 mM).
4

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
J. Kolinska and A. Grzelakowska
tion and emission spectra in the presence of various concentrations
intensity was also observed. However, the addition sodium bisul-
fite to solution of 5 results in the highest increase of fluorescence
intensity only at 410 nm. The changes in color of fluorescence of
compound 5 in phosphate buffer (10% CH3CN, pH 7.4) after the
addition of amino acids containing sulfhydryl group and bisulfite
under UV lamp are shown in Fig. S5.
Furthermore, similar results were observed for aliphatic thiol
compounds, such as 2-mercaptoethanol (ME) and thioglycolic acid
(TGA) (Fig. S6).
bisulfite (in the range of 0–40
lM) were registered (Fig. 2). After
the addition of HSOꢁ3 the absorption band located in the visible
range of the spectrum gradually undergoes a hipsochromic shift
and an increase in fluorescence intensity is observed.
According to the titration plot, the lowest concentration of
sodium bisulfite that can be determined using the presented com-
pound 5 was estimated to be 0.25 mM and it is comparable with the
values for other probes described in literature so far [19,39–41].
Moreover, in order to confirm that the optical response toward
thiol amino acid is the results of the reaction of the probe with sulf-
hydryl group present in the structure of amino acid, the emission
spectra of compound 5 in the presence of L-Cys which was previ-
ously treated with thiol masking reagent (NEM) were recorded.
As shown in Fig. S7, the styrylbenzimidazolium derivative in the
presence L-Cys pretreated with NEM did not enhance fluorescence
intensity at 410 nm.
3.3. Determination of the reaction responsible for bisulfite recognition
To verify the proposed sensing mechanism shown in Fig. 3A, the
product of the reaction of HSOꢁ3 with compound of 5 was con-
firmed by hydrogen nuclear magnetic resonance (1H NMR) spec-
troscopy. As shown in Fig. 3B, the signal at 8.02 ppm (Ha),
corresponding to the double bond in dicyanovinyl moiety of com-
pound 5, disappeared and two new signals at 4.38 and 4.33 ppm
The results indicate the probe 5 can be used to detect of bisulfite
and is able to distinguish HSOꢁ3 from other reactive sulfur species.
ꢁ
(Hb, Hc) appeared. Moreover, the adduct of 5-HSO3 was further
confirmed by HRMS analysis (m/z: [M + Na]+ calcd. for C19H14N4O3-
NaS 401.0684, found 401.0679) (Fig. S4).
3.5. Selectivity and interference for the detection of bisulfite
The addition of bisulfite to compound 5 induces the highest flu-
orescence intensity increase. To further assess the selectivity of
compound 5 for HSOꢁ3 , the competition tests were conducted.
The changes of fluorescence intensity ratios (I/I0) in the presence
of bisulfite after the addition of various amino acids (L-Cys, L-
Hcy, L-GSH, L-ACC, Gly, L-Glu, L-Lys), reactive sulfur species (SHꢁ,
Sꢁ2 ) and H2O2 were monitored (Fig. 7). As shown, the addition of
other competitive analytes does not affect the fluorescence
response of compound 5 to bisulfite. It can therefore be concluded
that the probe 5 can be successfully used for the detection of
sodium bisulfite over other amino acids.
3.4. Studies of the fluorescent response to various amino acids and
reactive sulfur species
The chromophore system attached to a dicyanovinyl group can
also be used as fluorescent probe to amino acids containing sulfhy-
dryl group [23–25]. Therefore, to check the selectivity, absorption
spectra and fluorescence emission spectra of compound 5 upon
the addition of various amino acids, namely of thiol amino acids,
L-cysteine (L-Cys), L-homocysteine (L-Hcy), L-glutathione (L-
GSH), N-acetyl-L-cysteine (L-ACC), in phosphate buffer (10% CH3-
CN, pH 7.4) were measured and are shown in Fig. 4. Additionally,
in Fig. 5 compound 5 towards representative anions, including
reactive sulfur species (NaSH, Na2S) and reactive oxygen species
(H2O2) at the same conditions are shown. In the presence of amino
acid and other compound tested, some changes in the absorption
spectra were observed. These changes were observed after about
30 min upon the addition the thiol amino acids (Fig. 6A-C). In
the case of sodium bisulfite the change was observed immediately
(Fig. 6D). It is worth that, almost complete disappearance of the
band ca. 400 nm and the highest absorbance value of the band
formed ca. 346 nm was observed for the bisulfite.
3.6. pH-dependence in the detection of thiol amino acids and bisulfite
As is well known, pH is also an important factor, which signifi-
cantly affects fluorescence measurements and the application of
fluorescent probes. Therefore, the investigation of the influence
of pH on the detection of bisulfite and thiol amino acid by probe
5 was performed. As shown in Fig. 8A, the fluorescence intensity
of the compound 5 with sodium bisulfite and thiol amino acids
at kmax 410 nm increased with increasing pH, reaching the highest
value at pH of 7.4. Above pH of 7.4, the decreased in fluorescence
intensity was observed for different studied analytes. As can be
seen in Fig. 8B, the fluorescence intensity of the compound 5 in
the presence of HSOꢁ3 at 410 nm was decreased, whereas at
550 nm was enhanced with the increase of pH (above 10). The
Fig. 4B and Fig. 5B illustrates the fluorescence spectra of 5 in the
presence of different amino acids and reactive sulfur species,
respectively. Upon the addition of thiol amino acids and reactive
sulfur species the formation a new maximum emission band
located at approximately 410 nm, but with a lower fluorescence
Fig. 2. (A) Absorption and (B) fluorescence spectra of compound 5 (10 mM) in the presence of different concentrations of HSOꢁ3 (0–40 mM) in phosphate buffer (10% CH3CN, pH
7.4).
5

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J. Kolinska and A. Grzelakowska
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
Fig. 3. (A) The proposed reaction of 5 for the determination of bisulfite anion. (B) Partial 1H NMR spectral change of 5 in the absence and in the presence of excess HSO3ꢁ in
dimethyl sulfoxide-d6.
Fig. 4. (A) Absorption and (B) fluorescence spectra changes of 5 (10 mM) in phosphate buffer (10% CH3CN, pH 7.4) after the addition of different amino acids (3 mM).
Fig. 5. (A) Absorption and (B) fluorescence spectra changes of 5 (10 mM) in phosphate buffer (10% CH3CN, pH 7.4) after the addition of different analytes (3 mM).
6

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
J. Kolinska and A. Grzelakowska
Fig. 6. Absorption spectra changes of 5 (10 mM) in phosphate buffer (10% CH3CN, pH 7.4) in the presence of amino acids containing thiol and bisulfite (3 mM) in time (0–
30 min.)
cytotoxicity of the tested styrylbenzimidazolium derivative toward
the cell line tested.
3.8. Application in wine samples
It is important to control the amount of bisulfite due to its wide
use in many products. The HSO3ꢁ anions are present in each wine,
which affect the product quality. To test the feasibility detecting
bisulfite in real samples using probe 5, the three type of wine were
selected. Based on the experimental results presented above, the
fluorescence response of the compound 5 toward bisulfite in these
samples was investigated. As shown in Table 2, the HSOꢁ3 levels in
the three wine samples were range from 3.88 to 7.17 mM. The
recovery values were in the range of 90.78%-99.67% in various
wine samples. It indicates that the probe 5 can be potential used
for the detection of bisulfite in real samples.
4. Conclusion
Here, the synthesis as well as detailed spectroscopic characteri-
zation of novel styrylbenzimidazolium-based fluorescent probe
with dicyanovinyl group as the recognition site for the detection
Fig. 7. The fluorescence intensity changes of compound 5 (10 mM) in the presence
of bisulfite after the addition of different competitor (3 mM) in phosphate buffer
(10% CH3CN, pH 7.4) (1. blank, 2. L-Cys, 3. L-Hcy, 4. L-GSH, 5. L-ACC, 6. Gly, 7. L-Glu,
of bisulfite are described. Upon reaction with bisulfite a strong flu-
orescence signal at 410 nm was generated. The probe reacts with
bisulfite via nucleophilic addition and it was confirmed by 1H
NMR measurements. The limit of detection for bisulfite was esti-
mated to be 0.25 mM. Moreover, the probe 5 has been successfully
implemented for the detection of bisulfite in various wine sample.
Additionally, this compound have slight cytotoxicity for the human
cells line.
8. L-Lys, 9. NaHS, 10. Na2S, 11. H2O2).
results indicate that the probe 5 can be used for the detection of
HSOꢁ3 under physiological conditions.
3.7. Cytotoxicity of styrylbenzimidazolium derivative
An important feature of new derivative that can be potentially
used in biological systems, is the low toxicity. Therefore, cytotoxi-
city of the novel styrylbenzimidazolium derivative was performed
using a Prestoblue-based assay. To this end, the human cells line
Based on the observed reactivity of the probe 5, we conclude
that it can be used for the detection of bisulfite, but its ability to
report thiols cannot be neglected. However, bisulfite reacts with
the probe faster than other reactive sulfur species. The presented
novel styrylbenzimidazolium-based fluorescent probe is favored
by its high reactivity toward bisulfite with the formation of the
product that exhibit strong fluorescence signal.
(HeLa) were treated with various concentrations (0-18.75
lmol/
L) of the compound 5. More than 70% of HeLa cells survived after
incubation with compound 5 (Fig. S8). That indicate the slight
7

´
J. Kolinska and A. Grzelakowska
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120151
Fig. 8. (A) pH effect on the fluorescence intensity at kmax of compound 5 (10 mM) with and without thiol amino acids and sodium bisulfite (3 mM) in phosphate buffer (10%
CH3CN, pH 7.4) (B) pH-dependent fluorescence spectra of compound 5 (10 mM) with HSOꢁ3 (3 mM) in phosphate buffer (10% CH3CN, pH 7.4).
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chemical characteristics, Food Chem. 348 (2021), https://doi.org/10.1016/
Results for the determination of HSOꢁ3 in three wine samples.
j.foodchem.2020.128922 128922.
Samples
HSOꢁ3 level
(mM)
HSOꢁ3 added
(mM)
HSOꢁ3 found
(mM)
Recovery %
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Rose wine 1
Red wine 2
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5.35
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´
Jolanta Kolinska: Conceptualization, Methodology, Investiga-
tion, Visualization, Writing - original draft. Aleksandra Grze-
lakowska: Methodology, Investigation, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Appendix A. Supplementary data
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