A rapid and visible colorimetric fluorescent probe for benzenethiol flavor detection

Article abstract of DOI:10.1016/j.foodchem.2019.02.033

Benzenethiols are a class of flavoring ingredients used in the food, pharmaceutical, cosmetics and chemical industries. A rapid and visible colorimetric fluorescent probe was developed for the detection of benzenethiol flavors. It provides rapid quantitative detection of benzenethiols at low levels, down to a limit of 10 nM. Test paper containing the probe changes color according to benzenethiol concentration (from colorless to pink, visible with the naked eye). The probe was also successfully used to test benzenethiol concentrations in real food samples. This study demonstrates that this novel probe can be employed as a benzenethiol testing tool.

Full text of DOI:10.1016/j.foodchem.2019.02.033

Accepted Manuscript
Analytical Methods
A rapid and visible colorimetric fluorescent probe for benzenethiol flavor de-
tection
Hao Wang, Xiaoming Wu, Shaoxiang Yang, Hongyu Tian, Yongguo Liu,
Baoguo Sun
PII:
S0308-8146(19)30347-4
DOI:
https://doi.org/10.1016/j.foodchem.2019.02.033
FOCH 24344
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2018
22 January 2019
7 February 2019
Please cite this article as: Wang, H., Wu, X., Yang, S., Tian, H., Liu, Y., Sun, B., A rapid and visible colorimetric
fluorescent probe for benzenethiol flavor detection, Food Chemistry (2019), doi: https://doi.org/10.1016/
j.foodchem.2019.02.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A rapid and visible colorimetric fluorescent probe for
benzenethiol flavor detection
Hao Wang, Xiaoming Wu, Shaoxiang Yang*, Hongyu Tian, Yongguo Liu and
Baoguo Sun
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Key laboratory of Flavor Chemistry, Beijing Technology and Business University,
No.11 Fucheng Road, Haidian District, Beijing 100048, People’s Republic of China
* Corresponding author. Telephone: +86-10-68985382. E-mail:
yangshaoxiang@th.btbu.edu.cn (S. X. Yang)
1

Abstract: Benzenethiols are a class of flavoring ingredients used in the food,
pharmaceutical, cosmetics and chemical industries. A rapid and visible colorimetric
fluorescent probe was developed for the detection of benzenethiol flavors. It provides
rapid quantitative detection of benzenethiols at low levels, down to a limit of 10 nM.
Test paper containing the probe changes color according to benzenethiol
concentration (from colorless to pink, visible with the naked eye). The probe was also
successfully used to test benzenethiol concentrations in real food samples. This study
demonstrates that this novel probe can be employed as a benzenethiol testing tool.
Keywords: Benzenethiol flavors; visible colorimetric; fluorescent probe
Highlights:
Development of a new, rapid, visible colorimetric fluorescent probe for benzenethiols.
The detection limit of this fluorescent probe is 10 nM.
Benzenethiol was successfully detected in real samples using the new probe.
2

1. Introduction
Benzenethiols are a class of flavoring ingredients with similar chemical properties
(Chinese Standards for Food Additives, 2015; Liu, & Sun, 2018). There are widely
used in the food, pharmaceutical, cosmetics and chemical industries (Jung, Chen, Kim,
& Yoon, 2013). Benzenethiols are recognized by the Flavor and Extract
Manufacturers Association (FEMA) as being “generally recognized as safe”, and
include benzenethiol (FEMA 3616) (Oser, & Ford, 1979), o-toluenethiol (FEMA
3240) (Hall, & Oser, 1970), 2-ethylthiophenol (FEMA 3345) (Oser, & Ford, 1973),
2,6-dimethylthiophenol (FEMA 3666) (Oser, Ford, & Bernard, 1984) and
2-mercaptoanisole (FEMA 4159) (Smith, et al., 2003). The odors of benzenethiol,
o-toluenethiol and 2-mercaptoanisole are considered meaty, but ethylthiophenol and
2,6-dimethylthiophenol are sulfurous. The odor description of benzenethiol is meaty,
sulfuraceous, phenolic, rubbery and allicious (Oser, & Ford, 1979), while
o-toluenethiol is meaty, smoked, sausage-like, smoky and phenolic (Hall, & Oser,
1970). 2-ethylthiophenol is sulfurous, roasted, smoky and meaty (Oser, & Ford, 1973),
2,6-dimethylthiophenol is sulfurous, roasted, meaty, metallic and phenolic (Oser, Ford,
& Bernard, 1984), and 2-mercaptoanisole is meaty, smoked, sausage-like, smoky, and
phenolic (Smith, et al., 2003). They are primarily useful for the preparation of onion,
garlic, meat and nut fragrances. The maximized survey-derived daily intake of
benzenethiol is 0.73 μg/person/day (Oser, & Ford, 1979), while that of o-toluenethiol
is 17.00 μg/person/day (Hall,
0.00012 μg/person/day (Oser,
&
Oser, 1970), 2-ethylthiophenol is
&
Ford, 1973), 2,6-dimethylthiophenol is
1.30 μg/person/day (Oser, Ford, & Bernard, 1984), and 2-mercaptoanisole is
1.50 μg/person/day (Smith, et al., 2003). Excessive amounts of benzenethiols can
induce central nervous system damage, vomiting, muscular weakness, coma, and even
death (Amrolia, Sullivan, Stern, & Munday, 1989; Chen, Tang, & Lin, 2016; Pagidi,
Kalluvettukuzhy, & Thilagar, 2018; Xiong, et al., 2017). The lethal dose (LD50) of
o-toluenethiol for mice is 100 mg/kg (Fairchild, & Stokinger, 1959; Meek, & Pettit,
1985). The LD50 of 2,6-dimethylthiophenol for rats is 3150 mg/kg (EFSA Panel on
Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), 2012).
The LD50 of benzenethiol is 46 mg/kg for rats (Fairchild, & Stokinger, 1958),
24 mg/kg for birds and 25 mg/kg for mice (Schafer, 1972). The LD50 of
2-mercaptoanisole is 1560 mg/kg for mice and 1740 mg/kg for rats (Ioannides,
Delaforge, & Parke, 1981). Hence, it is of great significance to develop a sensitive,
3

high selectivity, simple and rapid method of detecting benzenethiol flavors.
Until now, many methods for benzenethiol detection have been developed, such as
gas chromatography (GC) (Beiner, Popp, & Wennrich, 2002), high performance liquid
chromatography (HPLC) (Sun, et al., 2016) and nanomaterial-based sensors (Dreyer,
et al., 2011; Zhao, et al., 2011). Recently, fluorescent detection has attracted interest
due to its simplicity, high sensitivity, short response time and ease of observation
(Chan, Dodani, & Chang, 2012; Collot, et al., 2018; Denis, et al., 2018; Wang, et al.,
2018b). The first fluorescent probe for recognizing thiophenols was reported in 2007
based on a nucleophile substitution reaction mechanism (Jiang, et al., 2007). Up to
now, many thiophenol fluorescent probes have been reported (Chen, Zhou, Peng, &
Yoon, 2010; Guo, et al., 2018; Hong, Xia, Feng, & Feng, 2018; Lin, et al., 2017; Ma,
et al., 2016; Wang, et al., 2018a; Yao, et al., 2018), but most have been designed and
used for biological imaging. A fluorescent probe for benzenethiol flavors has not yet
been reported. Most thiophenol probes still have shortcoming; for example, they may
be unable to detect low concentrations of thiophenols due to having low sensitivity,
slow fluorescent turn-on response and lack of obvious color changes, and have slow
response speeds. Thus, there is a high demand for colorimetric fluorescent probes that
can rapidly detect benzenethiol flavors with high sensitivity.
In order to find a more sensitive and visible colorimetric benzenethiol flavor
fluorescent
(E)-6-(3-(4-nitrophenyl)-3-oxoprop-1-enyl)naphthalen-2-yl
2,4-dinitrobenzenesulfonate (Probe 1) is reported in this work. It uses
(4-nitrophenyl)-3-oxoprop-1-enyl)naphthalene as the fluorophore, and
probe,
a
new
probe
based
on
2,4-dinitrobenzenesulfonate as the reaction group. Probe 1 shows responsive and
visible colorimetrics for benzenethiol with the naked eye, and has a low detection
limit for benzenethiol flavors. In particular, the Probe 1 test paper reacts with different
concentrations of benzenethiol to produce observable color changes. Hence, the Probe
1 test paper could be employed as a powerful tool for the detection of benzenethiol
flavors.
2. Materials and methods
2.1 Chemicals
The chemicals 2-bromo-4’-nitroacetophenone (99%), trimethylamine (99%),
triphenylphosphine
(99%),
6-hydroxy-2-naphthaldehyde
(98%),
2,4-dinitrobenzenesulfonyl chloride (98%), glutathione (GSH, 98%), hydrazine
4

hydrate (N2H4, 99%), cysteine (Cys, 99%), benzenethiol (PhSH, 99%),
2,6-dimethylthiophenol (DMTP, 99%), 2-methylbenzenethiol (2-MBT, 99%), allyl
mercaptan (AMCT, 98%), mercaptopropionic acid (MCTP, 98%) and trimethylamine
(99%) were purchased from Bailing Wei (Beijing, China). The analytically pure
reagents sodium sulfide (Na2S), MgCl2 (magnesium chloride), KCl (potassium
chloride), sodium bromide (NaBr), sodium dihydrogen phosphate (NaH2PO4), sodium
chloride (NaCl), sodium sulfite (Na2SO3), sodium fluoride (NaF), sodium hydrogen
sulfite (NaHSO3), sodium sulfate (Na2SO4), potassium iodide (KI), tetrahydrofuran
(THF), N,N-dimethylformamide (DMF), methanol (CH3OH) and ethanol (C2H5OH)
were obtained from Innochem Science and Technology Co., Ltd. (Beijing, China).
2.2 Instruments
Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker AV 300
MHz NMR machine. High resolution mass spectrometer (HRMS) spectra were
implemented on a Bulu Ke Apex IV Fourier-Transform Mass Spectrometer (FTMS).
Fluorescence spectra were obtained on a Rili F-4600 fluorescence spectrometer.
2.3 Preparation of probe 1
As illustrated in scheme 1, 2-bromo-4’-nitroacetophenone (Compound 1; 4.88 g, 20
mmol) and triphenylphosphine (Compound 2; 5.24 g, 20 mmol) were dissolved in
tetrahydrofuran (50 mL). The mixture was heated to reflux for 4 h, then cooled to
25 °C to obtain compound 3. NaOH (2 M, 20 mL), methanol (24 mL) and H2O (28
mL) were added. The mixture was stirred at 25 °C overnight, then Compound 4 (a
yellow solid) was obtained by filtration.
6-Hydroxy-2-naphthaldehyde (Compound 5; 1.00 g, 5.8 mmol) and trimethylamine
(0.61 g) were dissolved in DMF (10 mL), then placed in an ice bath.
2,4-dinitrobenzenesulfonyl chloride (Compound 6; 3.09g, 11.6 mmol) in DMF (10
mL) was slowly added. The reaction mixture was stirred for 3 h at 40 °C. The
precipitate was collected by filtration, and then recrystallized from ethanol to give
Compound 7. Then, Compound 7 (0.50 g, 1.2 mmol) and Compound 4 (0.63 g, 1.5
mmol) were dissolved in tetrahydrofuran (25 mL), refluxed for 8 h, distilled and
recrystallized from CHCl3 to obtain Probe 1 (0.32 g, Scheme 1).
2.4 Preparation of solutions of probe 1 and analytes
Ethanol, as a reagent, was used to dissolve Probe 1, PhSH, Cys, DMTP, GSH,
AMCT, MCTP and 2-MBT. After mixing, a Probe 1 stock solution was obtained. The
analytes Na2S, MgCl2, KCl, NaBr, NaH2PO4, NaCl, Na2SO3, NaF, NaHSO3, Na2SO4,
5

KI and N2H4 were dissolved in distilled water to obtained 10 mM aqueous solutions.
Various concentrations could be obtained by diluting these stock solutions with
distilled water (Wang, et al., 2018b).
2.5 Preparation of real-world samples
Mineral water (bottle, 500 mL), coca cola (bottle, 500 mL), orange juice (bottle,
500 mL) and ham sausage (150 g) were purchased from wumei supermarket (Beijing,
P.R. China). The solid ham sausage sample was accurately quantified (100.00 g), then
chop up, ultrasonic extraction with ethanol (50 mL), filtered, concentration under
vacuum to 1 mL. A 20 μL sample solution was used for the determination. Different
concentrations of benzenethiol were added, and the 511 nm fluorescence signals of
samples were recorded.
2.6 The procedures of benzenethiols determination
Preparation of the test system: 0.02 mL probe solution was added to a cuvette. Then,
ethanol was poured into the cuvette to make up a volume of 2 mL. Finally, ion
solution was added. After waiting a few minutes, it was mixed completely. Samples of
mixture were analyzed in the fluorescence spectrometer using the conditions of λex =
356 nm, λem = 511 nm, temperature = 25 °C, voltage = 700 V, and slit widths = 5 nm, 5
nm.
3. Results and Discussion
3.1 Probe synthesis
Probe 1 was synthesized by a four-step reaction (Scheme 1). First, the intermediate
wittig reagent (Compound 4) was prepared by nucleophile substitution and
elimination reactions. Second, intermediate Compound 7 was obtained from a
nucleophile substitution reaction between Compounds 5 and 6. Lastly, Probe 1 was
created from a wittig reaction of Compounds 4 and 7. This synthetic process and
purification by recrystallization are classic organic chemistry methods. Probe 1 used
13C NMR, HRMS and 1H NMR for confirmation (Figs. A1-A3).
1H NMR (300 MHz, DMSO), δ (ppm): 9.15 (d, J = 2.2 Hz, 1H), 8.59 (dd, J = 8.7, 2.3
Hz, 1H), 8.43 (d, J = 14.0 Hz, 5H), 8.33 – 7.79 (m, 8H), 7.38 (dd, J = 8.9, 2.4 Hz, 1H);
13C NMR (75 MHz, DMSO), δ (ppm): 191.57, 154.09, 137.37, 133.83, 132.74,
130.63, 128.89, 128.65, 124.55, 122.19, 120.65, 119.75, 116.85; HRMS (ESI): calcd.
for [M-H]+ 548.039441, found 548.039739.
3.2 Sensing properties of Probe 1 towards benzenethiol
The benzenethiol flavors have similar chemical characteristics. Benzenethiol
6

(PhSH), 2,6-dimethylthiophenol (DMTP) and 2-methylbenzenethiol (2-MBT) were
obtained for this work. Benzenethiol (PhSH) was selected as a typical benzenethiol
flavor for testing the sensing properties of Probe 1 towards benzenethiol flavors.
The fluorescent response of Probe 1 to PhSH at different pHs from 3.0 to 10.0 was
investigated (Fig. 1a). The fluorescent intensity of Probe 1 did not change from pH
3.0 to 7.4, but increased from pH 7.4 to 10.0. However, as PhSH was added, the
fluorescent intensity of Probe 1 increased rapidly from pH 3.0 to 5.0, then stayed
almost constant from pH 5.0 to 7.4, before increasing again from pH 7.4 to 10.0. The
fluorescence intensity showed the greatest difference between Probe 1 and Probe
1-PhSH at pH 7.4; hence, pH-neutral conditions have the advantage of stabilizing
benzenethiols. Therefore, pH 7.4 was selected as the most suitable condition for the
subsequent experiments.
The fluorescence response of Probe 1 (10 μM) to different concentrations of PhSH
(0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM) was verified in 10 mM phosphate buffer
saline (PBS; pH 7.4) with ethanol (v:v = 1:3) at 25 °C (Fig. 1b). As different
concentrations of PhSH were added, the fluorescence intensity increased almost 2.5
times. The fluorescence signal at 511 nm increased constantly until 90 s, showing that
Probe 1 only needs 90 s to respond to PhSH. The results suggest that Probe 1 is
suitable for use for the rapid detection of PhSH.
The solution of Probe 1 in PBS (pH 7.4) with ethanol (v:v = 1:3) was added to
different concentrations of PhSH (0.0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM,
10 μM, 20 μM) and changes in fluorescence intensity were observed (Fig. 1c). The
intensity of the highest fluorescence emission peak at 511 nm increased with the
addition of PhSH (Fig. 1d). The fluorescence intensity was linearly correlated with
concentrations of PhSH ranging from 0–1 μM (Fig. 1e). The detection limit (LOD) of
Probe 1 for PhSH was 12 nM, based on LOD = 3 SD/B according to the definition of
IUPAC (LOD =3SD/B, SD represents standard deviation of the blank solution and B
is the slope of the linear regression) (Wang, et al., 2018b). These results suggest that
Probe 1 is an appropriate tool for the quantitative detection of PhSH at low
concentrations.
Benzenethiol could be detected by using paper strips containing Probe 1. Waterman
filter paper was cut into 1 cm × 1 cm pieces and dipped in a 1 mM DMSO solution of
Probe 1. The paper strips were then dried in a vacuum drying oven at 50 °C to rapidly
evaporate the solvent (Xiong, et al., 2017). The prepared test paper was then placed
7

on a glass plate. 20 μL of PhSH of different concentrations (0.00 μM, 5 μM, 10 μM,
50 μM, 100 μM,) were added. After 30 s, the color of the test paper changed
according to the concentration of PhSH, as observed by the naked eye under natural
light or 254 nm UV light (Fig. 1f). All results indicate that Probe 1 can be applied as a
sensor for the rapid detection of PhSH at concentrations of 0.00 mM, 0.05 mM, 0.10
mM, 0.50 mM and 1.00 mM.
To explore the detection sensitivity of Probe 1 for PhSH, various compounds were
-
2-
tested, including H2S, HSO3 , N2H4, GSH, H2O2, Mg2+, Na+, K+, SO4 , Cl-, F-, Br-, I-,
2-
-
SO3 , H2PO4 , Cys, allyl mercaptan, mercaptopropionic acid, methylbenzenethiol and
2,6-dimethylthiophenol. As shown in Figure 1g, these competitors exhibited limited
fluorescence responses, except for AMCT, 2-MBT and DMTP. The competitors
MCTP, H2S, GSH and Cys, which contain a sulfhydryl group, had no effect on PhSH.
At the same time, competition experiments were conducted by adding PhSH to Probe
1 solutions containing the above competitors. The fluorescence response of Probe 1
was similar for the PhSH and PhSH + competitor samples. The fluorescence emission
intensity of Probe 1 (10 μM) to 2-MBT was similar with PhSH, the fluorescence
emission intensity of AMCT and DMTP was almost 10% less than PhSH. This
indicates that Probe 1 is highly selective for benzenethiols.
2,6-dimethylthiophenol (DMTP) and 2-methylbenzenethiol (2-MBT) have similar
chemical characteristics to PhSH (Liu, & Sun, 2018). Reaction times, and the
relationship between fluorescence intensity and DMTP and 2-MBT concentrations
using Probe 1, were examined at the same time. The fluorescence response of Probe 1
(10 μM) to different concentrations of DMTP (0.1μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8
μM) was verified in 10 mM phosphate buffer saline (PBS; pH 7.4) in ethanol at 25 °C
(Fig. 2a). The results show that Probe 1 needs 320 s to respond to DMTP. The
fluorescence response of Probe 1 (10 μM) to different concentrations of 2-MBT (0.2
μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM) was verified under the same conditions (Fig.
2b). The results show that Probe 1 needs 75 s to respond to 2-MBT. The reaction rates
of PhSH, DMTP and 2-MBT with Probe 1 were ranked 2-MBT > PhSH > DMTP. As
this reaction is a nucleophile substitution reaction, the reaction rate is determined by
the nucleophiles of the thiol group. Methyl groups are electron donating, such that the
nucleophilicity of 2-MBT was increased. However, due to the steric effect, the
nucleophilicity of DMTP was decreased.
The intensity of the highest fluorescence peak at 511 nm increased with addition of
8

DMTP (Fig. 2c) and 2-MBT (Fig. 2d). The fluorescence intensity was linearly related
to the concentration of DMTP in the range 0–0.8 μM (Fig. 2e), and the LOD of Probe
1 for DMTP was 15 nM. A linear relationship between 2-MBT concentration and
fluorescence intensity was found in the 0–1.0 μM range (Fig. 2f), and the LOD of
Probe 1 for 2-MBT was 7 nM. These results suggest that Probe 1 is a wonderful tool
for the quantitative detection of low levels of DMTP and 2-MBT.
3.3 Reaction mechanism
A possible response mechanism may contribute to the thiolysis of the 2,
4-dinitrobenzenesulfonyl group of Probe 1 via a nucleophile substitution reaction that
generates Compound 8, (2,4-dinitrophenyl) (phenyl)sulfane (Compound 9) and SO2
(Compound 10), as shown in Scheme 2 (Wang, et al., 2018b). To verify this response
mechanism, Probe 1’s reaction with PhSH was analyzed by MS. A peak at m/z =
318.30 was observed, which correlates with the formation of Compound 8 (Fig. A4).
A peak at m/z = 277.36 correlates with the formation of Compound 9 (Fig. A5). The
results suggest that the mechanism by which Probe 1 senses PhSH is most likely
based on the thiolysis of the 2, 4-dinitrobenzenesulfonyl moiety.
3.4 Detection of PhSH in real samples
The results thus far indicate that Probe 1 has good recognition for PhSH in complex
environments. The ability of Probe 1 to detect PhSH in real samples was
demonstrated to prove its applicability. Four kinds of real samples (mineral water;
Coca Cola; orange juice; ham sausage; 20 μL) were added to Probe 1 solutions (10
μM). Then, different amounts of PhSH (0.2 μM and 0.4 μM) were added due to the
detection limit of Probe 1 was 10 nM and the linear ranges were from 0–1 μM. As
shown in Table 1, amounts of 0, 0, 0 and 0.0618 μM PhSH were found in the four
kinds of real samples (mineral water; Coca Cola; orange juice; ham sausage). Hence,
Probe 1 could detect PhSH in these four real samples. The recovery values of 94.40 %
to 101.0 % show that Probe 1 has good ability to detect PhSH in real samples (mineral
water; Coca Cola; orange juice; ham sausage). The results show that Probe 1 is a
feasible and practical testing method for detecting PhSH in real samples. As DMTP,
2-MBT, 2-ETP and 2-MP have similar chemical characteristics to PhSH, Probe 1
could be expected to detect concentrations of DMTP, 2-MBT, 2-ETP and 2-MP in
real samples.
Most existing thiolphenol fluorescent probes have been designed and used for
biological imaging (Chen, Zhou, Peng, & Yoon, 2010; Guo, et al., 2018; Hong, Xia,
9

Feng, & Feng, 2018; Lin, et al., 2017; Ma, et al., 2016; Wang, et al., 2018a; Yao, et al.,
2018). A fluorescent probe for benzenethiol flavor detection has not been reported
before. Probe 1 is a novel fluorescent probe that is very easy to synthesize. Paper
strips containing Probe 1 changed color in 30 s when exposed to different
concentrations of PhSH. These changes could be observed by the naked eye under
natural light or 254 nm UV light. In addition, this visual change shows that Probe 1
can be used to develop a naked eye detection tool for the detection of PhSH.
Furthermore, Probe 1 was successfully used to detect PhSH concentrations in real
samples.
4. Conclusions
In summary, a rapid and visible colorimetric fluorescent probe for detecting PhSH
was developed. The function of Probe 1 relies on the nucleophile substitution reaction
of Probe 1 with PhSH, which generates (2,4-dinitrophenyl)(phenyl)sulfane, as verified
by MS. Probe 1 is a rapid and useful quantitative tool for detecting benzenethiol
flavors in low concentrations. Importantly, Probe 1 test paper reacted with different
concentrations of PhSH to produce different colors, changing gradually from colorless
to pink. This indicates that Probe 1 could be employed as a testing tool for PhSH.
Furthermore, our work shows that Probe 1 is useful for testing PhSH levels in
real-world samples.
Acknowledgements
Thank you for the Support Project of High-level Teachers in Beijing Municipal
Universities in the Period of 13th Five–year Plan (CIT&TCD201804021), Scientific
and technological innovation and service capacity building - basic research business
funding - study on the flavor of typical Chinese traditional dishes
(PXM2018-014213-000033).
Appendix A. Supplementary data
HRMS, 13C NMR and 1H NMR spectra of probe 1; MS spectra of compounds 8 and 9.
Declaration of Interest Statement
Declarations of interest: none. The authors declare no competing financial interest.
10

References
Amrolia, P., Sullivan, S. G., Stern, A., & Munday, R. (1989). Toxicity of aromatic thiols in the
human red blood cell. Journal of Applied Toxicology, 9(2), 113−118.
Beiner, K., Popp, P., & Wennrich, R. (2002). Selective enrichment of sulfides, thiols and
methylthiophosphates from water samples on metal-loaded cation-exchange materials for gas
chromatographic analysis. Journal of Chromatography A, 968(1-2), 171−176.
Chan, J., Dodani, S. C., & Chang, C. J. (2012). Reaction-based small-molecule fluorescent probes
for chemoselective bioimaging. Nature Chemistry, 4(12), 973.
Collot, M., Fam, T. K., Ashokkumar, P., Faklaris, O., Galli, T., Danglot, L., et al. (2018).
Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in
Cells and Tissues. Journal of the American Chemical Society, 140(16), 5401−5411.
Chinese Standards for Food Additives - GB2760-2015.
Chen, H., Tang, Y., & Lin, W. (2016). Recent progress in the fluorescent probes for the specific
imaging of small molecular weight thiols in living cells. TrAC Trends in Analytical
Chemistry, 76, 166−181.
Chen, X., Zhou, Y., Peng, X., & Yoon, J. (2010). Fluorescent and colorimetric probes for detection
of thiols. Chemical Society Reviews, 39(6), 2120−2135.
Dreyer, D. R., Jia, H. P., Todd, A. D., Geng, J., & Bielawski, C. W. (2011). Graphite oxide: a
selective and highly efficient oxidant of thiols and sulfides. Organic & biomolecular
chemistry, 9(21), 7292−7295.
Denis, M., Pancholi, J., Jobe, K., Watkinson, M., & Goldup, S. M. (2018). Chelating Rotaxane
Ligands as Fluorescent Sensors for Metal Ions. Angewandte Chemie International Edition,
57(19), 5310−5314.
EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF). (2012).
Scientific Opinion on Flavouring Group Evaluation 08, Revision 5 (FGE. 08Rev5): Aliphatic
and alicyclic mono-, di-, tri-, and polysulphides with or without additional oxygenated
functional groups from chemical groups 20 and 30. EFSA Journal, 10(7), 2837.
Fairchild, E. J., & Stokinger, H. E. (1958). 1. Acute Toxicity of Some Aliphatic and Aromatic
Thiols (Mercaptans). American Industrial Hygiene Association Journal, 19(3), 171−189.
Fairchild, E. J., & Stokinger, H. E. (1959). Toxicologic Studies on Organic Sulfur Compounds. 1.
Acute Toxicity of Some Aliphatic and Aromatic Thiols (Mercaptans). Journal of Occupational
and Environmental Medicine, 1(1), 67.
Guo, S. H., Leng, T. H., Wang, K., Wang, C. Y., Shen, Y. J., & Zhu, W. H. (2018). A colorimetric
and turn-on NIR fluorescent probe based on xanthene system for sensitive detection of
thiophenol and its application in bioimaging. Talanta, 185, 359−364.
Hall, R. L., & Oser, B. L. (1970). Recent progress in the consideration of flavoring ingredients
under the Food Additives Amendment. 4. GRAS substances. Food Technology, 24(5), 25−34.
Hong, J., Xia, Q., Feng, W., & Feng, G. (2018). A dicyanoisophorone-based near-infrared
11

fluorescent probe and its application for detecting thiophenols in water and living cells. Dyes
and Pigments, 159, 604−609.
Ioannides, C., Delaforge, M., & Parke, D. V. (1981). Safrole: its metabolism, carcinogenicity and
interactions with cytochrome P-450. Food and Cosmetics Toxicology, 19, 657−666.
Jiang, W., Fu, Q., Fan, H., Ho, J., & Wang, W. (2007). A highly selective fluorescent probe for
thiophenols. Angewandte Chemie, 119(44), 8597−8600.
Jung, H. S., Chen, X., Kim, J. S., & Yoon, J. (2013). Recent progress in luminescent and
colorimetric chemosensors for detection of thiols. Chemical Society Reviews, 42(14),
6019−6031.
Liu, H. L., & Sun, B. G. (2018). Effect of fermentation processing on the flavor of Baijiu. Journal
of Agricultural and Food Chemistry, 2018, 66(22), 5425−5432.
Lin, L., Zeng, X., Shen, Y., Zhu, H., & Qian, Y. (2017). An ultrasensitive fluorescent probe for
rapid determination of thiophenols. Talanta, 165, 321−325.
Ma, Q., Xu, J., Zhang, X., Zhou, L., Liu, H., & Zhang, J. (2016). A naphthalene-based two-photon
fluorescent probe for selective and sensitive detection of thiophenols. Sensors and Actuators B:
Chemical, 229, 434−440.
Meek, J., & Pettit, B. (1985). Avoidable accumulation of potentially toxic levels of benzothiazoles
in babies receiving intravenous therapy. The Lancet, 326(8464), 1090−1092.
Oser, B. L., & Ford, R. A. (1973). Recent progress in the consideration of flavoring ingredients
under the Food Additives Amendment. 6. GRAS substances. Food Technology, 27(1), 64−67.
Oser, B. L., & Ford, R. A. (1979). Recent progress in the consideration of flavoring ingredients
under the Food Additives Amendment. 12. GRAS substances. Food Technology, 33(7), 65−73.
Oser, B. L., Ford, R. A., Bernard, B. K. (1984). Recent progress in the consideration of flavoring
ingredients under the Food Additives Amendment. 13. GRAS substances. Food Technology,
38(10), 66−89.
Pagidi, S., Kalluvettukuzhy, N. K., & Thilagar, P. (2018). Triarylboron anchored luminescent
probes: Selective detection and imaging of thiophenols in the intracellular
environment. Langmuir, 34(28), 8170−8177.
Schafer, E. W. (1972). The acute oral toxicity of 369 pesticidal, pharmaceutical and other
chemicals to wild birds. Toxicology and Applied Pharmacology, 21(3), 315−330.
Smith, R. L., Cohen, S. M., Doull, J., Feron, V. J., Goodman, J. I., Marnett, L. J., et al. (2003). The
22nd publication by the FEMA Expert Panel presents safety and usage data on 185 new
generally recognized as safe flavoring ingredients and describes an approach to assessing the
safety of natural flavor complexes. 22. GRAS substances. Food Technology, 59(8), 24−62.
Sun, Y., Lv, Z., Sun, Z., Wu, C., Ji, Z., & You, J. (2016). Determination of thiophenols with a
novel fluorescence labelling reagent: analysis of industrial wastewater samples with SPE
extraction coupled with HPLC. Analytical and Bioanalytical Chemistry, 408(13), 3527−3536.
Wang, K., Zhao, C. X., Leng, T. H., Wang, C. Y., Lu, Y. X., Shen, Y. J., et al. (2018a). Dual
12

quenching strategy for sensitive detection of toxic thiolphenols based on a NIR-illuminant
platform with a large Stokes shift. Dyes and Pigments, 151, 194−201.
Wang, H., Wang, J. L., Yang, S. X., Tian, H. Y., Liu, Y. G., & Sun, B. G. (2018b). Highly selective
and rapidly responsive fluorescent probe for hydrogen sulfide detection in wine. Food
Chemistry, 257, 150−154.
Xiong, L., Ma, J., Huang, Y., Wang, Z., & Lu, Z. (2017). Highly Sensitive Squaraine-Based
Water-Soluble Far-Red/Near-Infrared Chromofluorogenic Thiophenol Probe. ACS sensors, 2(4),
599−605.
Yao, Z., Ge, W., Guo, M., Xiao, K., Qiao, Y., Cao, Z., et al. (2018). Ultrasensitive detection of
thiophenol based on a water-soluble pyrenyl probe. Talanta, 185, 146−150.
Zhao, W., Liu, W., Ge, J., Wu, J., Zhang, W., Meng, X., et al. (2011). A novel fluorogenic hybrid
material for selective sensing of thiophenols. Journal of Materials Chemistry, 21(35),
13561−13568.
Lists of Figures and Tables
Scheme 1. Synthesis of Probe 1.
Scheme 2. The mechanism for reaction of Probe 1 with PhSH.
Figure 1. (a) Fluorescent responses of Probe 1 (10 μM) added to PhSH (300 μM) in
buffer solution with pHs of 3–10). Tests were performed in triplicate. (b)
Time-dependent fluorescence spectra of Probe 1 (10 μM) in the presence of PhSH
(0.2, 0.4, 0.6, 0.8, 1.0 μM) in PBS (pH 7.4) with ethanol (v/v, 1:3) at 25 °C.
Tests were performed in triplicate. (c) Fluorescence spectra of Probe 1 (10 μM) with
PhSH (0, 0.2, 0.4, 0.6, 0.8, 1.0, 10, 20 μM). (d) Fluorescence intensity of Probe 1 (10
μM) with PhSH (0, 0.2, 0.4, 0.6, 0.8, 1.0, 10, 20 μM). (e) Plot of fluorescence
intensity differences with PhSH amounts of 0 to 1.0 μM. (f) Photograph of Probe 1
paper strips subjected to PhSH (0, 5, 10, 50, 100 μM) under ambient light and 254 nm
UV light. (g) Fluorescence intensity change of Probe 1 (10 μM) upon addition of
-
various species (10 μM for each. 1, blank; 2, H2S; 3, HSO3 ; 4, N2H4; 5, GSH; 6, H2O2;
2-
2-
-
7, Mg2+; 8, Na+; 9, K+; 10, SO4 ; 11, Cl-; 12, F-; 13, Br-; 14, I-; 15, SO3 ; 16, H2PO4 ;
17, Cys; 18, 2-MBT; 19, DMTP; 20, AMCT; 21, MCTP. 10 μM for PhSH).
Tests were performed in triplicate.
Figure 2. (a) Time-dependent fluorescence spectra of Probe 1 (10 μM) in the presence
of DMTP (0.1, 0.2, 0.4, 0.6, 0.8 μM) in PBS (pH 7.4) with ethanol (v/v, 3:1) at 25 °C.
13

Tests were performed in triplicate. (b) Time-dependent fluorescence spectra of Probe
1 (10 μM) in the presence of 2-MBT (0.2, 0.4, 0.6, 0.8, 1.0 μM) in PBS (pH 7.4) with
ethanol (v/v, 3:1) at 25 °C. Tests were performed in triplicate. (c) Fluorescence spectra
of Probe 1 (10 μM) with DMTP (0, 0.1, 0.2, 0.4, 0.6, 0.8 μM). (d) Fluorescence
spectra of Probe 1 (10 μM) with 2-MBT (0, 0.2, 0.4, 0.6, 0.8, 1.0 μM). (e) Plot of
fluorescence intensity differences with DMTP ranging from 0 to 0.8 μM. (f) Plot of
fluorescence intensity difference with 2-MBT ranging from 0 to 1.0 μM. Tests were
performed in triplicate.
Table 1. Determination of benzenethiol concentrations in real samples
Scheme 1.
14

Scheme 2.
15

Figure 1.
17

Figure 2.
18

Sample
PhSH amount PhSH amount PhSH amount Recovery
s
originally
added
(μM)
detected
(μM)
present (μM)
0.2000
0.4000
0.2000
0.4000
0.2000
0.4000
0.2000
0.4000
0.2020
0.4012
0.2064
0.3996
0.1924
0.3817
0.2506
0.4595
101.0%
100.3%
103.2%
99.90%
96.20%
95.43%
94.40%
99.43%
0.0061
0.0037
0.0046
0.0038
0.0057
0.0028
0.0038
0.0045
Mineral water
Coca Cola
0.0000
0.0000
0.0000
0.0618
Orange juice
Ham sausage
Tests were performed in triplicate. s, standard deviation.
Table 1.
19

Highlights:
Development of a new, rapid, visible colorimetric fluorescent probe for benzenethiols.
The detection limit of this fluorescent probe is 10 nM.
Benzenethiol was successfully detected in real samples using the new probe.
20