The construction of an effective far-red fluorescent and colorimetric platform containing a merocyanine core for the specific and visual detection of thiophenol in both aqueous medium and living cells

Article abstract of DOI:10.1039/c9nj03020d

Thiophenol (PhSH) is toxic to the environment and biological systems although it is an indispensable material in the synthetic processes of chemical products. In this work, we designed and synthesized a malonitrile-based colorimetric and deep-red emission fluorescent dosimeter specifically for thiophenol detection by switching on the intramolecular charge transfer process. The 2,4-dinitrobenzene moiety in the probe could be cleaved by thiophenol, which led to strong deep-red fluorescence increase at 645 nm (50-fold) with a low detection limit of 9 nM. The maximum plateau of emission intensity was reached in about 30 min after the addition of thiophenol. Simultaneously, the presence of thiophenol could result in a prominent color change from orange to blue. Furthermore, the dosimeter could quantitatively sense thiophenol spiked in water samples and successfully visualize thiophenol in living SH-SY5Y cells.

Full text of DOI:10.1039/c9nj03020d

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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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DOI: 10.1039/C9NJ03020D
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Construction of an effective far-red fluorescent and colorimetric
platform containing a merocyanine core for specific and visual
detection of thiophenol in both aqueous medium and living cells
Received 00th January 20xx,
Accepted 00th January 20xx
Xin Sun,a Mengzhao Wang,a Yanan Lu,a Zhengliang Lu,*,a Chunhua Fan,*,a Yizhong Lu*,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
Thiophenol (PhSH) is toxic to environment and biological system although it is an indispensable material in synthetic
processes of chemical products. In this work, we design and synthesize a malonitrile-based colorimetric and deep-red
emission fluorescent dosimeter specifically for thiophenol detection by switching on the intramolecular charge transfer
process. The 2,4-dinitrobenzene moiety in the probe can be cleaved off by thiophenol, which leads to a strong deep-red
fluorescence increase at 645 nm (50-fold) with a low detect limit of 9 nM. The maximum plateau of emission intensity was
reached in about 30 min since addition of thiophenol. Simultaneously, the presence of thiophenol can result in a
prominent colorimetric change from orange to blue. Furthermore, the dosimeter can quantitatively sense thiophenol
sparked in water samples and successfully visualize thiophenol in living SH-SY5Y cells.
pioneering work by Wang,17 considerable amount of effort has been
spent to construction of fluorescent probes sensing thiophenol,
1. Introduction
which usually contain a recognition unit such as benzenesulfonyl or
phenol tailed by two nitro groups.18-29 The strong nucleophilic
attack of thiolate anions can readily cleave off the protected groups
and finally release fluorescence through modulation of electron
processes.30-40 Moreover, as we know, far-red/near-infrared
fluorescent dyes have many advantages such deep tissue
penetration, low photodamage and photobleaching to biology and
low interference from background.41-43 It is to be noted that there
have been only a few of thiophenol probes showing far-red
fluorescence or even near-infrared emission (NIR) in literatures.44-50
In 2014, Feng’s group reported a thiophenol probe emitting
fluorescence at 720 nm containing dicyanomethylene-4H-
benzopyran core with a limit of detection (LOD) of 0.15 μM in PBS
buffer-DMSO (7:3, v/v).47 In 2017, a fluorescent probe with
emission at 623 nm for thiophenol was developed by conjunction of
coumarin anchored by phenothiazine with a LOD of 2.9 nM in PBS
buffer assisted with CTAB (1mM).48 Zhu’s group developed another
NIR fluorescent probe as a dicyanoisophorone derivative for
thiophenol with the detection limit of 0.42 μM in PBS buffer-DMSO
(1:1, v/v).49 Recently, Song’s group successfully discriminatorily
detected thiophenol, hydrogen sulfide and mercapto amino acids
with one fluorescent probe in a triple-emission mode.50 Notably,
these excellent probes still have some drawbacks such as use of
highly proportional organic solvent, assistance of CTAB, or high
detection limits. Especially, lack of far-red/NIR fluorophores
extremely encumbered further development of performance-
perfect probes and potential application in biology.
It is well known that thiophenol is highly toxic to organisms and
extremely detrimental to environment although they are
extensively used in organic synthesis as an indispensable raw
material for preparing various agrochemicals, pharmaceuticals and
others.1-3 Due to high toxicity and carcinogenicity, trace inhalation
or dermal contact with thiophenol might damage the central
nervous system and cause several disorders, including but not
limited to respiratory disease, headache, nausea, vomit, muscular
weakness, etc.4 Furthermore, reactive superoxide radicals as well as
hydrogen peroxide probably could be generated through oxidation
from thiophenol to disulphide, which might cause severe oxidative
damage to erythrocyte cells.5 It is reported that the thiophenol
concentration from 0.01 to 0.4 mM to fish and from 2.15 to 46.2
mg/kg in mouse was reported as the median lethal dose,6, 7 so that
thiophenol is regarded as one of the top pollutants. Apparently, it is
of a high demand to develop specifically sensing methods for
thiophenol in environment and biology.
In this end, compared with traditional methods including
electrochemistry and UV-vis spectrometry, small-molecule based
fluorescence imaging has been attracting more and more attention
due to their simplicity, easy operation, on-spot detection, low cost,
non-destruction, and high spatiotemporality.8-16 Since the
a School of Chemistry and Chemical Engineering, University of Jinan, Jinan
250022, China. Email: zhengliang.lu@yahoo.com; chm_fanch@ujn.edu.cn
b School of Materials Science and Engineering, University of Jinan, Jinan 250022,
China. Email: mse_luyz@ujn.edu.cn
As we know, a practical way to construct the far-red/NIR
fluorophores is introduction of suitable electron-withdrawing
groups (EWGs) which can effectively modulate charge/electron
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [NMR spectra of the
compounds, MS and chromatograms of the reaction systems, supplementary
fluorescent spectra, cytotoxicity assay]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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transfer processes of targeted probes.51-53 Furthermore, besides the nucleophilicity of thiophenol could easily break down VaineweAtrhtieclre bOonlninde
54, 55
DOI: 10.1039/C9NJ03020D
shift of excitation/emission wavelength to the NIR range, this kind between the 2,4-dinotrophenyl group and the fluorophore.
of EWGs also improves their sensitivity or selectivity toward With this thought in mind, as displayed in Scheme 1, a deep-red
analytes in biology. With this thought, we envisioned that a simple fluorescent FRP-Thio for PhSH was synthesized in the presence of
malonitrile group could substantially ameliorate chemical and/or triethylamine in CH2Cl2 through linking the 2,4-dinotrophenyl unit
photophysical properties of probes. Therefore, in this study, we and the fluorophore FRP-OH. This fluorophore tethered by the
developed
a
thiophenol fluorescent probe (FRP-Thio) as
a
strong electron-withdrawing cyano groups emits the deep-red
merocyanine derivative tailed by malonitrile with the 2,4- fluorescence due to the excellent intramolecular charge transfer
dinitrophenol group. The probe showed a colorimetric change and process (ICT). Therefore, the presence of thiophenol could
also far-red emission (50-fold increase) by selectively and sensitively effectively cut off the ether bond, which finally released the
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detecting thiophenol with a LOD of 9 nM. Benefited by the fluorophore FRP-OH and led to
malonitrile group extending
a
dramatic fluorescent
1
a
π-conjugation, the emission enhancement. FRP-Thio was characterized by H NMR, 13C NMR as
wavelength of FRP-Thio was moved into the far-red fluorescence well as HRMS. (Fig. S1-S3).
range. In addition, FRP-Thio could successfully visualize thiophenol
in aqueous solution and living SH-SY5Y cells.
Scheme 1. The synthesis of FRP-Thio.
2. Experimental
2.1. Synthesis of FRP-Thio
FRP-OH (0.14 g, 0.5 mmol) was dissolved in 20 mL of MeCN and
refluxed after addition of K2CO3 (0.09 g, 0.6 mmol) as well as 1-
fluoro-2,4-dinitrobenzene (0.112 g, 0.6 mmol) with stirring
overnight. The title compound FRP-Thio as a red solid was purified
by flash chromatography using ethyl acetate/petroleum ether (1/1,
1
v/v) (0.13 g，58%). H NMR (600 MHz, DMSO-d6) δ 8.94 (d, J = 2.8
Hz, 1H), 8.52 (dd, J = 9.2, 2.8 Hz, 1H), 8.23 (s, 1H), 7.62 (d, J = 8.5 Hz,
1H), 7.49 – 7.47 (m, 1H), 7.45 (d, J = 9.2 Hz, 1H), 7.37 (s, 1H), 7.18
(dd, J = 8.5, 2.4 Hz, 1H), 2.75 (t, J = 6.0 Hz, 2H), 2.65 (t, J = 6.1 Hz,
2H), 1.77 (p, J = 6.1 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 158.37,
156.38, 153.92, 153.51, 151.16, 142.86, 140.55, 130.76, 130.21,
129.67, 129.04, 122.38, 121.67, 119.27, 117.50, 116.91, 116.09,
110.63, 107.64, 70.17, 28.63, 24.64, 20.32. HRMS [C23H14N4O6+H+]
m/z: calcd. 443.0991, found 443.0979.
2.2. Fluorescent imaging in SH-SY5Y cells
SH-SY5Y cells were grown in DMEM containing 10% fetal bovine
serum as well as 1% penicillin and streptomycin. SH-SY5Y cells
adhered for 24 h before subsequent experiments. Two groups of
cell experiments were set up. In a control group, SH-SY5Y cells were
only pretreated with 10 μM FRP-Thio for 30 min. In the second
group, SH-SY5Y cells were first incubated with 100 μM thiophenol
and then further with10 μM FRP-Thio in turn.
Fig. 1. UV-vis absorption (a), fluorescence (b) and color (Inset of b)
changes of FRP-Thio (10 μM) in the absence and presence of
thiophenol (200 μM) in DMF-H2O (2/8, v/v, 20 mM PBS buffer, pH
7.4; λex/em = 560/645 nm).
3.2. UV-Vis absorption and fluorescence spectra
3. Results and discussion
We firstly checked both the absorption and fluorescence spectra
of FRP-Thio probe (10 μM) were performed in 20% DMF aqueous
solution (v/v, 20 mM PBS buffer, pH 7.4) with or without
thiophenol. As Fig. 1a demonstrated, free FRP-Thio exhibited only a
broad absorption band at around 500 nm. However, Addition of
thiophenol (20 equiv) into the probe solution led to three strong
3.1. Design and synthesis of FRP-Thio
As mentioned before, the suitable tailing group in a push-pull
system could modulate the emission wavelengths of targeted
probes, and also significantly improve their selectivity and
sensitivity. Additionally, many studies confirmed that the
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absorption peaks at 528, 560, and 605 nm. As shown in Fig. 1b, the remarkable enhancement of fluorescence enhancement at 645 nm
free probe is nonfluorescent. However, addition of thiophenol excited at 560 nm. Apparently, this enhancement of emission
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DOI: 10.1039/C9NJ03020D
resulted in a distinct far-red fluorescence enhancement at 645 nm. intensity was reasonably attributed to the release of the
Furthermore, the probe FRP-Thio displayed remarkable colorimetric fluorophore emitting at 645 nm. With gradual addition of
responses from orange to blue-green with gradual addition of thiophenol, the emission at 645 nm sharply rose and finally reached
thiophenol under natural light (Inset of Fig. 1b). These outstanding a maximum plateau with thiophenol (20 equiv). Additionally, the
changes of color and spectra indicate FRP-Thio can be a promising fluorescence intensities at 645 nm was perfectly proportional to
tool for thiophenol by naked eyes even without any instruments thiophenol concentration (Fig. 2b). The LOD of 9.0 nM of FRP-Thio
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under physiological conditions.
for thiophenol is estimated. Gradual addition of thiophenol (0, 40,
80, 120, 160, 200 μM) induced the distinctly colorimetric change as
shown in Fig. 2c. Therefore, we can conclude that FRP-Thio can
specifically detect thiophenol by spectroscopic or/and colorimetric
methodology.
Response time is an important parameter for reaction-based
probes. The kinetic profile of FRP-Thio (10 μM) reacting with
thiophenol (200 μM) in 20% DMF PBS buffer (20 mM, pH 7.4) were
investigated to determine the response time. The fluorescence at
645 nm increased with addition of thiophenol (200 μM) at first (Fig.
S4). In about 30 minutes, it reached the maximum plateau.
Moreover, Addition of thiophenol (100 μM) did not prolong the
formation time of the maximum fluorescence intensity. No
difference of equilibrium time for two different concentrations
testified that the response time of FRP-Thio is not affected by the
concentration of thiophenol. There is no obvious decay of emission
intensity in the subsequent another 30 min. Hence, all these
experiments support that FRP-Thio can reliably and rapidly detect
thiophenol.
Fig. 3. Fluorescence responses at 645 nm of FRP-Thio (10 μM) with
500 μM relevant species (1. Blank, 2. K+, 3. Na+, 4. Al3+, 5. Mg2+, 6.
Zn2+, 7. Ba2+, 8. Cu2+, 9. Fe2+, 10. Mn2+, 11. Cd2+, 12. Ca2+, 13. F–, 14.
Cl–, 15. SO42–, 16. SO32–, 17. AcO–, 18. ClO–, 19. H2O2, 20. Asp, 21.
Try, 22. Gly, 23. Val, 24. Ile, 25. Glu, 26. Arp, 27. Leu, 28. Ala, 29. His,
30. Lys, 31. Met, 32. Phe, 33. Tyr, 34. Ser, 35. Pro, 36. Thr, 37. H2S,
38. Hcy, 39. Cys, 40. GSH) as red bars or with following addition of
30 equiv of thiophenol (green bars) excited at 560 nm.
Fig. 2. (a) Fluorescence spectral changes at 645 nm of FRP-Thio (10
μM) upon addition of thiophenol (0-20 equiv) excited at 560 nm.
Each spectrum was recorded at 30 min after addition of thiophenol.
(b) The linear relationship between emission intensity at 645 nm
and PhSH concentration. (c) Color change of FRP-Thio (10 μM) upon
addition of thiophenol at different concentration (0, 40, 80, 120,
160, 200 μM) under natural light.
The subsequent titration experiments were performed to disclose
the relationship between the probe (10 μM) toward thiophenol at
different concentration in 20% DMF PBS buffer (20 mM, pH 7.4). As
displayed in Fig. 2a, free FRP-Thio did not emit any obvious
3.3. Selectivity and anti-interference studies
To further evaluate the specificity, the competing experiments in
20% DMF PBS buffer (20 mM, pH 7.4) were investigated by
fluorescence. As expected, addition of thiophenol led to
a
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