sensors
Article
A Biocompatible Colorimetric TriphenylamineDicyanovinyl Conjugated Fluorescent Probe for
Selective and Sensitive Detection of Cyanide Ion
in Aqueous Media and Living Cells
Zi-Hua Zheng 1,2 , Zhi-Ke Li 3 , Lin-Jiang Song 3 , Qi-Wei Wang 1 , Qing-Fei Huang 1, *
and Li Yang 3, *
1
2
3
*
Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China;
[email protected] (Z.-H.Z.); [email protected] (Q.-W.W.)
University of Chinese Academy of Sciences, Beijing 100049, China
Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center,
Chengdu 610041, China; [email protected] (Z.-K.L.); [email protected] (L.-J.S.)
Correspondence: [email protected] (Q.-F.H.); [email protected] (L.Y.); Tel.: +86-28-8550-2157 (L.Y.)
Academic Editors: Jong Seung Kim and Min Hee Lee
Received: 12 December 2016; Accepted: 4 February 2017; Published: 19 February 2017
Abstract: A colorimetric and turn-on fluorescent probe 1 bearing triphenylamine-thiophene and
dicyanovinyl groups has been synthesized and used to detect cyanide anion via a nucleophilic
addition reaction. Probe 1 exhibited prominent selectivity and sensitivity towards CN− in aqueous
media, even in the presence of other anions such as S2− , HS− , SO3 2− , S2 O3 2− , S2 O8 2− , I− , Br− , Cl− ,
F− , NO2 − , N3 − , SO4 2− , SCN− , HCO3 − , CO3 2− and AcO− . Moreover, a low detection limit (LOD,
51 nM) was observed. In addition, good cell membrane permeability and low cytotoxicity to HeLa
cells were also observed, suggesting its promising potential in bio-imaging.
Keywords: biocompatible; cell imaging; colorimetric assay; dicyanovinyl; cyanide anion
1. Introduction
Anions play an important role in a wide range of chemical and biological processes. Given this,
anion recognition has been well studied in recent years [1]. Among various anions, cyanide anion
(CN− ) is a well-known anion that is toxic to living organisms [2]. As advocated by the World Health
Organization (WHO), the maximum concentration of cyanide anion in drinking water should be
1.9 µM [3]. However, CN− is widely released from industry waste, certain natural plants, cigarette
smoke, etc. [4], and can thus be a serious threat to people’s health and the environment. Hence,
developing effective quantitative determination methods for CN− is urgent and essential.
In order to quickly, easily and accurately detect cyanide anion, various analysis methods were
developed, including titrimetric [5], electrochemical decices [6–8], voltammetric [9], fluorometric [10–17]
and so on. Compared with other methods, fluorometric and colorimetric responses based on chemical
reactions have attracted much attention due to their simple operation, high selectivity, high sensitivity
and low cost [18–22]. The sensing mechanisms of fluorometric and colorimetric chemosenors for
detecting CN− have been reported over the past few years, including hydrogen bonding reactions [23–27],
forming cyanide anion complexes [28,29], electron transfer reactions [30–32] and nucleophilic addition
reactions [33–40]. Among them, the nucleophilic addition reactions generally have high selectivity for
detecting CN− .
Triphenylamine-based thiophene dicyanovinyl compounds, which contain an electron-donating
triphenylamine moiety and a strong electron-withdrawing dicyanovinyl group, were reported as
Sensors 2017, 17, 405; doi:10.3390/s17020405
www.mdpi.com/journal/sensors
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red-emitting fluorophores with large Stokes shifts [41]. In recent years, some fluorescent probes
with dicyanovinyl moieties for detecting cyanide anions have been reported [42–47]. Consequently,
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we hypothesized that triphenylamine-based thiophene dicyanovinyl compounds could be applied to
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2017,
17, 405
2 ofto
9
selectively
detect
cyanide
anion.
hypothesized
that triphenylamine-based
thiophene dicyanovinyl compounds could be applied
selectively
detect cyanide
With
this hypothesis
inanion.
mind, probe 1 was synthesized and exhibited prominent selectivity
hypothesized
that
triphenylamine-based
dicyanovinyl
compounds
could selectivity
be appliedand
to
With this
hypothesis
in mind,
probe
1thiophene
was
synthesized
and exhibited
prominent
and sensitivity
towards
cyanide
anion
with
significant
turn-on
fluorescent
response
in PBS/DMSO
selectively
detect
cyanide
anion.
towards
cyanide
anion with
significant
response and
in PBS/DMSO
(4/6,probe
(4/6, sensitivity
v/v,With
pH =this
7.4)
solution
(Scheme
1). Herein,
weturn-on
report afluorescent
dual
colorimetric
fluoresecent
hypothesis
in mind,
1 was
synthesized
exhibited and
prominent
selectivity
and
v/v, pH
= 7.4) solution
(Scheme
1).probe
Herein,
we report
a dualand
colorimetric
fluoresecent
probe
to
to selectively
and
sensitively
detect
cyanide
anion inturn-on
aqueous
media. Moreover,
low
cytotoxicity
and
sensitivity
towards
cyanide
anion
with
significant
fluorescent
response
in
PBS/DMSO
(4/6,
selectively and sensitively detect cyanide anion in aqueous media. Moreover, low cytotoxicity and
goodv/v,
cellpH
membrane
permeability
in
HeLa
cells
were
also
observed,
suggesting
its
promising
potential
= 7.4)
solution permeability
(Scheme 1). Herein,
we cells
report
a dual
fluoresecent
probe to
good cell
membrane
in HeLa
were
alsocolorimetric
observed, and
suggesting
its promising
in bio-imaging.
selectively
sensitively detect cyanide anion in aqueous media. Moreover, low cytotoxicity and
potential inand
bio-imaging.
good cell membrane permeability in HeLa cells were also observed, suggesting its promising
potential in bio-imaging.
Scheme 1. The sensing mechanism of probe 1.
Scheme 1. The sensing mechanism of probe 1.
Scheme 1. The sensing mechanism of probe 1.
2. Results and Discussion
2. Results and Discussion
2.
Results
andofDiscussion
2.1.
Synthesis
Probe 1
2.1. Synthesis of Probe 1
As shown
in Scheme
2, probe 1 was synthesized via a 3-step synthesis route according to a
2.1. Synthesis
of Probe
1
literature
[41]. The
structure
of probe
1 was fully
by NMR
andaccording
ESI-MS to
As
shownmethod
in Scheme
2, probe
1 was
synthesized
via acharacterized
3-step synthesis
route
As shownDetailed
in Scheme
2, probe
1 was
synthesized
via a 3-step synthesis
route
according
to a
spectroscopy.
synthetic
process
and
structure
characterization
are
given
in
the
Experimental
a literature method [41]. The structure of probe 1 was fully characterized by NMR and ESI-MS
literature
method
[41].
The
structure
of
probe
1
was
fully
characterized
by
NMR
and
ESI-MS
section and
in the Electronic
Supplementary
Information
(ESI).
spectroscopy.
Detailed
synthetic
process
characterization
given
in Experimental
the Experimental
spectroscopy.
Detailed
synthetic
processand
andstructure
structure characterization
areare
given
in the
section
and and
in the
Electronic
Supplementary
(ESI).
section
in the
Electronic
SupplementaryInformation
Information (ESI).
N
N
2
a
a
N
Br
N
Br
3
b
b
c
N
S
c
N
4
S
CN
N
S
CHO
CHO
N
S
1
CN
CN
CN
Scheme
(a) NBS, DMF, r.t., 99%1 yield; (b) (54
2 2. The synthesis of 3probe 1; Reagents and Conditions:
formylthiophen-2-yl) boronic acid, Pd(PPh3)4, dioxane, reflux, 3.5 h, 67% yield; (c) malononitrile,
Scheme
The
synthesis
of
probe
Reagents
and Conditions:
(a) NBS,(a)
DMF,
r.t.,DMF,
99% yield;
(b) (5-yield;
piperidine,
reflux,
3 h, 70%
Scheme
2. 2.
The
synthesis
ofyield.
probe1; 1;
Reagents
and Conditions:
NBS,
r.t., 99%
3)4, dioxane, reflux, 3.5 h, 67% yield; (c) malononitrile,
formylthiophen-2-yl)
boronic
acid,
Pd(PPh
(b) (5-formylthiophen-2-yl) boronic acid, Pd(PPh3 )4 , dioxane, reflux, 3.5 h, 67% yield; (c) malononitrile,
piperidine,over
reflux,
3 h,Anions
70% yield.
2.2. Selectivity
Other
piperidine, reflux, 3 h, 70% yield.
After the over
identification
of the optimal fluorescence measurement conditions, a series of relevant
2.2. Selectivity
Other Anions
and interfering
anions
were investigated to study the selectivity. Before the addition of various
2.2. Selectivity
over Other
Anions
After
identification
the optimal
fluorescence measurement
series
of relevant
anions,
thethe
light-pink
probeofsolution
is non-fluorescent
upon excited conditions,
at 370 nm. aThe
addition
of 10
After
the identification
of the
optimal
measurement
conditions,
a series
of relevant
and
interfering
were
investigated
study
selectivity.
Before
the addition
of various
2−to
−, SO3the
2−, S
equivalents
of 16anions
representative
anions (Sfluorescence
, HS
2O32−, S2O82−, I−, Br−, Cl−, F−, NO2−, N3−, SO42−,
anions,
the
light-pink
probe
solution
is
non-fluorescent
upon
excited
at
370
nm.
The
addition
of 10
and interfering
anions
were
investigated
to
study
the
selectivity.
Before
the
addition
of
various
anions,
−
−
−
2−
SCN , HCO3 , CO3 and AcO ) did not cause significant fluorescence changes (Figure 1a), whereas
2−, HS−, SO32−, S2O32−, S2O82−, I−, Br−, Cl−, F−, NO2−, N3−, SO42−,
equivalents
of
16
representative
anions
(S
−
the light-pink
probe
solution
is non-fluorescent
upon excited
at 370
nm.and
The
of 10intensity
equivalents
the color of
the probe
1 solution
faded in the presence
of CN (10
equiv.)
theaddition
fluorescence
−, HCO3−, CO32− and AcO
2−−,)remarkable.
2− , Ssignificant
2− S fluorescence
2− I− , Brchanges
− , Cl
− whereas
− , SO
2−
did−not
(Figure
1a),
−,
of 16 SCN
representative
HS
, SOcause
F− , NO
enhancement
at anions
480 nm(S
was
good
of− ,probe
1 toward
3 Based
2 O3on ,the
2O
8 , selectivity
2 , N3 CN
4 ,
− (10 equiv.) and the fluorescence intensity
the
color
of
the
probe
1
solution
faded
in
the
presence
of
CN
2− and AcO
experiments
were−further
explored.
As shown influorescence
Figure 1b, the
fluorescence
intensity
of
SCN−competition
, HCO3 − , CO
) did not
cause significant
changes
(Figure
1a), whereas
3
enhancement
480 nm at
was
remarkable.
Based
on the good
selectivity
of probe
1 toward
CN−,
− at
−
probe
+
CN
remained
a
high
level
by
the
addition
of
other
interfering
anions
(10
equiv.),
the color of the probe 1 solution faded in the presence of CN (10 equiv.) and the fluorescence intensity
competition
experiments
wereoffurther
As−shown
in Figure
1b, the fluorescence intensity of
suggestingatthat
response
probe 1explored.
toward
CN
good
anti-interference
enhancement
480thenm
was remarkable.
Based
onhas
the
good
selectivity ofability.
probe 1 toward CN− ,
probe + CN− remained at a high level by the addition of other interfering anions (10 equiv.),
competition
experiments
were further
As− shown
inanti-interference
Figure 1b, the fluorescence
intensity of
suggesting
that the response
of probeexplored.
1 toward CN
has good
ability.
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probe + CN− remained at a high level by the addition of other interfering anions (10 equiv.), suggesting
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2017, 17, 405
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that the
response
of probe 1 toward CN− has good anti-interference ability.
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(a) Fluorescent
responses
probe11 (5
(5 µM)
µM) towards
anions
(50 (50
µM)µM)
in PBS/DMSO
FigureFigure
1. (a)1.Fluorescent
responses
ofofprobe
towardsvarious
various
anions
in PBS/DMSO
(4/6, pH = 7.4) solution. λex = 370 nm, Slits: 2.5 nm/5 nm; (b) Competing responses of probe 1 (5 µM)
(4/6, pH = 7.4) solution. λex = 370 nm, Slits: 2.5 nm/5 nm; (b) Competing responses of probe 1 (5 µM)
at 480 nm towards various analytes (50 µM) in PBS/DMSO (4/6, pH = 7.4) solution. Black bar, probe
at 480 nm towards various analytes (50 µM)
in PBS/DMSO (4/6, pH = 7.4) solution. Black bar, probe
and probe + anions; red bar, probe + CN− + anions. λex = 370 nm, Slits: 2.5 nm/5 nm.
− + 1anions.
Figure
1. (a) Fluorescent
responses
of probe
(5 µM) towards
various
in PBS/DMSO
and probe
+ anions;
red bar, probe
+ CN
λex = 370
nm,anions
Slits:(50
2.5µM)
nm/5
nm.
(4/6, pH = 7.4) solution. λex = 370 nm, Slits: 2.5 nm/5 nm; (b) Competing responses of probe 1 (5 µM)
2.3. Colorimetric
Fluorescent
Detection
at 480 nm and
towards
various analytes
(50 µM) in PBS/DMSO (4/6, pH = 7.4) solution. Black bar, probe
2.3. Colorimetric
and Fluorescent
Detection
and probe
+ anions; red bar,
probe + CN− + anions. λex = 370 nm, Slits: 2.5 nm/5−nm.
The UV-vis spectra changes of probe 1 (5 µM) upon addition of CN (50 µM) in PBS/DMSO (4/6,
− (50
pH =UV-vis
7.4) solution
canchanges
be clearlyofobserved
S1)upon
and the
absorption
at 505
nmµM)
decreased.
The
The
spectra
probe 1(Figure
(5 µM)
addition
of CN
in PBS/DMSO
2.3. Colorimetric
and Fluorescent
Detection
free
probe
1
exhibits
absorption
at
505
nm
with
a
pink
color
because
of
ICT
process
in
the
large
π(4/6, pH = 7.4) solution can be clearly observed (Figure S1) and the
absorption at 505 nm decreased.
− (50 µM) in PBS/DMSO (4/6,
The UV-vis
spectra changes
of probe 1assay
(5 µM)
upon
addition of
conjugation.
Subsequently,
a colorimetric
was
performed.
AsCN
shown
in Figure 2a, among tested
The free pH
probe
1 solution
exhibitscanabsorption
at 505 nm with
pink
color because
of ICT process
= 7.4)
be clearly observed
S1) a
and
the absorption
at 505 nm
Thein the large
analytes,
only
cyanide anion
resulted in (Figure
an obvious
color
change from
light decreased.
pink to colourless.
π-conjugation.
Subsequently,
a colorimetric
assay
was
performed.
Asprocess
shown
in Figure
free probe
1 exhibits absorption
at 505 nm with
a pink
color
because of ICT
in the
large π- 2a, among
Moreover, a bright blue light was achieved in the solution of probe 1 with CN− under the irradiation
conjugation.
Subsequently,
a colorimetric
assay
was
performed.color
As shown
in Figure
2a,light
among
tested
testedofanalytes,
only
cyanide
anion
resulted
in
an
obvious
change
from
pink
to colourless.
a UV-lamp at 365 nm, which is visible by the naked eye (Figure 2b).
analytes, only cyanide anion resulted in an obvious color change from light pink−to colourless.
Moreover, a bright blue light was achieved in the solution of probe 1 with
CN
under
the
irradiation
Moreover, a bright blue light was achieved in the solution of probe 1 with CN− under the irradiation
of a UV-lamp
at 365 at
nm,
is visible
naked
eye
(Figure
of a UV-lamp
365which
nm, which
is visibleby
by the
the naked
eye
(Figure
2b). 2b).
Figure 2. Fluorescence images of probe 1 (5 µM) upon addition of various anions (50 µM) in
PBS/DMSO
pH = 7.4)images
solution
light
(a); addition
and 365 nm
UV lamp
(b). (50 µM) in
Figure 2. (4/6,
Fluorescence
of under
probe visible
1 (5 µM)
upon
of various
anions
Figure 2.PBS/DMSO
Fluorescence
images of probe 1 (5 µM) upon addition of various anions (50 µM) in PBS/DMSO
(4/6, pH = 7.4) solution under visible light (a); and 365 nm UV lamp (b).
(4/6,
pH = 7.4)
solution
under1 visible light (a); and 365 nm UV lamp (b).
2.4. Spectral
Titration
of Probe
2.4. Spectral Titration of Probe 1
The fluorescence titration experiment was performed by adding different concentrations of
2.4. Spectral
Titration
of Probetitration
1 0 toexperiment
fluorescence
performed
byof
adding
concentrations
of was
cyanideThe
anion
ranging from
75 µM to was
a 5 µM
solution
probedifferent
1 (Figure
S3). Saturation
cyanide anion ranging from 0 to 75 µM to a 5 µM solution of probe 1 (Figure S3). Saturation was
observed
when the titration
concentration
of cyanidewas
anion was raised to
µM. As
shown inconcentrations
Figure 3a, a
The
fluorescence
experiment
by62.5
adding
different
of
observed
when the concentration
of cyanide anionperformed
was raised to 62.5
µM. As shown
in Figure 3a, a
concentration-dependent fluorescence enhancement was observed when −CN− from 0 to 25 µM was
cyanide anion
ranging
from
0
to
75
µM
to
a
5
µM
solution
of
probe
1
(Figure
S3).
Saturation
was
concentration-dependent fluorescence enhancement was observed when CN from 0 to 25 µM was
added at 480 nm. A good linear relationship between the fluorescence intensity and the concentration
at 480
A good linear relationship
between
fluorescence
concentration
observedadded
when
thenm.
concentration
of cyanide
anionthewas
raised intensity
to 62.5 and
µM.theAs
shown in Figure 3a,
− could be obtained in the range of 0 to 25 µM (R2 = 0.99698).
of CN
− could be obtained in the range of 0 to 25 µM (R2 = 0.99698).
of CN
−
a concentration-dependent
fluorescence
enhancement
was
observed
when
CN
from
0 to 25
µM was
To To
calculate
the
(LOD),the
thestandard
standard
deviation
of the
blank
measurements
calculate
thelimit
limitofofdetection
detection (LOD),
deviation
of the
blank
measurements
was was
addeddetermined
atdetermined
480 nm.byAby
good
linear
between
the 1fluorescence
intensity
theequation
concentration
measuring
the
fluorescent
intensity
probe
1 10
times
[48–50].
Based
onequation
the
measuring
therelationship
fluorescent intensity
ofofprobe
10
times
[48–50].
Based
on and
the
− could be obtained in the range of 0 to 25 µM (R2 = 0.99698).
of CN(LOD
= 3σ/m)
and
theLOD
LODwas
was
calculated
to 51
benM,
51 nM,
which
is much
(LOD
= 3σ/m)
andthe
thefluorescence
fluorescence titration,
titration, the
calculated
to be
which
is much
lower
than
the
maximum
level of
of cyanide
cyanide
anion
µM)
in in
drinking
water
by the
lower
than
the
maximum
level
anion
(1.9
µM)
drinking
water
permitted
byWHO,
the WHO, was
To
calculate
the
limit
of detection
(LOD),
the(1.9
standard
deviation
ofpermitted
the
blank
measurements
suggesting
promisingpotential
for the
detection
levels
anion
in water
samples.
suggesting
itsits
promising
for
theintensity
detectionof
oflow
low
levels
oftimes
cyanide
anion
in
water
samples.
determined
by measuring
thepotential
fluorescent
of
probe
1 of
10cyanide
[48–50].
Based
on
the equation
(LOD = 3σ/m) and the fluorescence titration, the LOD was calculated to be 51 nM, which is much
lower than the maximum level of cyanide anion (1.9 µM) in drinking water permitted by the WHO,
suggesting its promising potential for the detection of low levels of cyanide anion in water samples.
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Figure
(a) Fluorescence
spectra
probe11(5(5µM)
µM) in
in the
the presence
concentration
of CN
Figure
3. (a)3.Fluorescence
spectra
ofofprobe
presenceofofvarious
various
concentration
of CN−
(0–25 µM) in PBS/DMSO (4/6, pH = 7.4) solution; (b) Linear relation between the fluorescent intensity
(0–25 µM) in PBS/DMSO (4/6, pH = 7.4) solution; (b) Linear relation between the fluorescent intensity
−
the range of 0–25 µM. λex = 370 nm, Slits: 2.5 nm/5 nm.
at 480 nm and the concentration of CN
− in
−
at 480Figure
nm and
theFluorescence
concentration
of CN
in the
ofthe
0–25
µM. λex
370 nm,
Slits: 2.5 nm/5
3. (a)
spectra
of probe
1 (5range
µM) in
presence
of =
various
concentration
of CNnm.
−
(0–25 µM) in PBS/DMSO (4/6, pH = 7.4) solution; (b) Linear relation between the fluorescent intensity
2.5. The
Sensing Mechanism
at 480 nm
and the concentration of CN−in the range of 0–25 µM. λex = 370 nm, Slits: 2.5 nm/5 nm.
2.5. The Sensing
Mechanism
As proposed in the literature [51], the sensing mechanism of probe 1 based on a nucleophilic
1H-NMR
As
in
thebeen
literature
[51],bythe
sensing
mechanism
of probe
1 based
a nucleophilic
addition
reaction
has
confirmed
spectroscopy
(Figure
4). Thus,
to a 20on
mM
solution
2.5.proposed
The Sensing
Mechanism
1
of probe
1 in has
CDCl
3 cyanide
anion by
(0.5 H-NMR
equiv.) was
added at room
temperature
and
a new
proton
addition
reaction
been
confirmed
spectroscopy
(Figure
4).
Thus,
to
20
mM
solution
As proposed in the literature
[51], the sensing mechanism of probe 1 based on a nucleophilic
2a). When the amount of CN− was raised to 1.0 equiv., some significant
signal
appeared
at
4.5
ppm
(H
of probe
1
in
CDCl
cyanide
anion
(0.5
equiv.)
was
added
at
room
temperature
and
a
new
proton
signal
3 has been confirmed by 1H-NMR spectroscopy (Figure 4). Thus, to a 20 mM solution
addition reaction
1a, 7.73 ppm)
proton
signal
changes
were
observed.
For
example,
the
vinyl
proton
signal
(H
2a
−
appeared
at 4.5
ppm
(H3 cyanide
). Whenanion
the amount
of CN
was raised
to 1.0
equiv., some
proton
of probe
1 in
CDCl
(0.5 equiv.)
was added
at room
temperature
and significant
a new proton
disappeared gradually and a 2anew proton signal at 4.5 ppm
(H2a) was observed.
two proton
1a , 7.73The
− was raised
signalsignal
changes
wereatobserved.
example,
the
vinyl
proton
signal
(H
ppm)
disappeared
).1bWhen
the amount
of
CN
to
1.0
equiv.,
some
significant
appeared
4.5 ppm (HFor
signals on the thiophene ring (H , 7.66 ppm; H1c, 7.30
ppm) disappeared and were observed
around
2a ) was
protonand
signal
changes
were
observed.
For example,
the
vinyl proton
(H1a, 7.73
ppm)
gradually
a new
proton
signal
at 4.5 ppm
(H
observed.
The signal
two proton
signals
on the
1d) adjacent to the thiophene ring shifted from 7.49
7.00 ppm. In addition,
the
two
proton
signals
(H
2a) was observed. The two proton
1b
1c
disappeared
gradually
and
a
new
proton
signal
at
4.5
ppm
(H
thiophene
H , 7.30
ppm)
disappeared
and were
around
7.00
ppm toring
7.36(H
ppm., 7.66
Theseppm;
important
proton
signal
changes conformed
withobserved
the reference.
While
1.5ppm.
1dppm;
signals on
thetwo
thiophene
ring
(H1b, 7.66
H1c, 7.30
ppm)
disappearedring
andshifted
were observed
around
In addition,
the
proton
signals
(H
)
adjacent
to
the
thiophene
from
7.49
ppm
−
−
equiv. CN was added, the reaction accomplished1dcompletely. In addition, the [1 − CN] adduct was to
7.00 ppm. In addition, the two proton signals (H ) adjacent to the thiophene ring shifted from 7.49
7.36 ppm.
These important
signal changes
with at
them/z
reference.
equiv. CN−
characterized
by mass proton
spectrometry
analysis,conformed
and the peak
457.1471While
(calc.1.5
457.1492)
ppm to 7.36 ppm. These important
proton signal changes conformed with −the reference. While 1.5
−
corresponding
to [1 accomplished
− CN] was clearly
observed In
(Figure
S10). the [1 − CN] adduct was characterized
was added,
the reaction
completely.
addition,
equiv. CN− was added, the reaction accomplished completely. In addition, the [1 − CN]− adduct was
by mass
spectrometry
analysis,
and
the
peak
at
m/z
457.1471
(calc. 457.1492) corresponding to
characterized by mass spectrometry analysis, and the peak at m/z 457.1471 (calc. 457.1492)
−
[1 − CN]
was clearly
S10).
corresponding
to [1 observed
− CN]− was(Figure
clearly observed
(Figure S10).
Figure 4. 1H-NMR spectral changes of probe 1 (20 mM) upon addition of CN− (as
tetrabutylammonium salts) in CDCl3.
Figure
4. H-NMR spectral changes of probe 1 (20 mM) upon −
of CN (as
Figure
4. 1 H-NMR
spectral changes of probe 1 (20 mM) upon addition of CN addition
(as tetrabutylammonium
tetrabutylammonium salts) in CDCl3.
salts) in CDCl3 .
1
−
Sensors 2017, 17, 405
Sensors 2017, 17, 405
2.6. Cellular Imaging
5 of 10
5 of 9
2.6. Cellular Imaging
To explore its potential in bio-imaging, the cytotoxicity of probe 1 was investigated by a
To2017,
explore
potential
in bio-imaging,
probe 1 was
investigated
a standard
17, 405itsAs
5 ofwhen
9
standardSensors
MTT
assay.
shown
in Figure the
5, cytotoxicity
HeLa cellofviability
was
slightly by
reduced
the
MTT 2017,
assay.
As shown in Figure 5, HeLa cell viability was slightly reduced when the probe
Sensors
17, 405
5 of 9
probe concentration
was raised up to 40 µM, indicating acceptable cytotoxicity of probe 1.
concentration was raised up to 40 µM, indicating acceptable cytotoxicity of probe 1.
2.6. Cellular Imaging
2.6. Cellular Imaging
To explore its potential in bio-imaging, the cytotoxicity of probe 1 was investigated by a standard
Toassay.
exploreAs
itsshown
potential
bio-imaging,
thecell
cytotoxicity
probe
1 wasreduced
investigated
bythe
a standard
MTT
in inFigure
5, HeLa
viability of
was
slightly
when
probe
MTT
assay. Aswas
shown
5, HeLa
cell acceptable
viability was
slightlyofreduced
concentration
raisedinupFigure
to 40 µM,
indicating
cytotoxicity
probe 1. when the probe
concentration was raised up to 40 µM, indicating acceptable cytotoxicity of probe 1.
Figure 5. HeLa cell viability values (%) estimated by MTT assay at different concentrations of probe
Figure 5.1 HeLa
cell viability values (%) estimated by MTT assay at different concentrations of probe 1
for 24 h.
for 24 h.
Probe 1 was next applied to detect cyanide anion in living HeLa cells. As shown in Figure 6,
5. HeLa
cell viability values
estimated
MTT
at
different
concentrations
of probe
HeLaFigure
cells were
incubated
probe(%)
1 (10
µM) forby
1h
andassay
with
different
concentrations
of CN−
Probe 1 1was
appliedwith
to detect
cyanide
anion
inthen
living
HeLa
cells.
As shown
in Figure 6,
for 24next
h.
Figure
5. HeLa
cellµM;
viability
(%)D,estimated
by MTT
assay
atUnder
different
concentrations
of probe
(A, E:
0 µM;
B, F: 10
C, G:values
80 µM;
H: 160 µM)
for 15
min.
the
fluorescent microscope,
HeLa cells
were
incubated with probe 1 (10 µM) for 1 h and then with different concentrations of CN−
1 for 24 h.intracellular blue fluorescence was observed (Figure 6G–H). Moreover, the fluorescence
remarkable
Probe
1 was
applied
to detect
cyanide
anion
in 15
living
HeLa
cells. the
As shown
in Figure
6,
(A, E: 0 µM;
B, F:showed
10
µM;next
G: 80 µM;
D, H:
160manner.
µM)
for
min.
Under
microscope,
intensity
a C,
concentration-dependent
As shown
in Figure
7, fluorescent
HeLa cells were
HeLa cells were incubated with probe 1 (10 µM) for 1 h and then with different concentrations of CN−
Probe
1 wasdifferent
next
applied
to detectofcyanide
in (Figure
living
cells.
As
Figure
incubated
with
concentrations
probe
(0anion
µM; 2.5
µM;
5.0HeLa
µM;
10
µM)Moreover,
forshown
1 h andinthen
with6,
remarkable
intracellular
blue
fluorescence
was
observed
6G–H).
the
fluorescence
(A, −E: 0 µM; B, F: 10 µM; C, G: 80 µM; D, H: 160 µM) for 15 min. Under the fluorescent microscope, −
HeLa
cells
were
incubated
with
probe
1
(10
µM)
for
1
h
and
then
with
different
concentrations
of
CN
(80
µM)
for
15
min.
The
fluorescence
intensity
also
showed
a
concentration-dependent
manner.
intensity remarkable
showed a intracellular
concentration-dependent
As shown
in Figure
7, HeLa
wereCN
incubated
blue fluorescence manner.
was observed
(Figure 6G–H).
Moreover,
thecells
fluorescence
− in
(A,
E: 0 µM; B,probe
F: 10 1µM;
C, G:the
80 potential
µM; D, H:
µM)
for
15vitro
min.cellular
Under systems
the fluorescent
microscope,
In summary,
showed
to160
detect
CN
in a concentration−
with different
concentrations
of probe (0 µM; −2.5 µM;
5.0 µM;
µM)inforFigure
1 h and
intensity
showed a concentration-dependent
manner.
As 10
shown
7, then
HeLa with
cells CN
were (80 µM)
remarkable
blueprobe
fluorescence
observed (Figure 6G–H). Moreover, the fluorescence
dependent intracellular
manner for both
and CN was
.
incubated
with different intensity
concentrations
probe (0 µM;
2.5 µM; 5.0 µM; 10 µM) for 1 h
and then In
with
for 15 min.
The fluorescence
alsoofshowed
a concentration-dependent
manner.
summary,
intensity
showed a concentration-dependent
manner.
As shown in Figure 7, HeLa
cells were
− (80 µM) for 15 min. The fluorescence −
CN
intensity
also
showed
a
concentration-dependent
manner.
in vitro
probe 1incubated
showed with
the potential
to detect CN
cellular
a concentration-dependent
different concentrations
of probe
(0 µM;
2.5 µM; systems
5.0 µM; 10in
µM)
for 1 h and then with
In summary, probe 1 showed
the potential to detect CN− in vitro cellular systems in a concentrationµM)
for 15and
min.CN
The−fluorescence
intensity
also
showed
a
concentration-dependent
manner.
mannerCN
for− (80
both
probe
.
dependent manner for both probe and CN−.
In summary, probe 1 showed the potential to detect CN− in vitro cellular systems in a concentrationdependent manner for both probe and CN−.
Figure 6. Images of living HeLa cells: HeLa cells incubated with probe 1 (10 µM) for 1 h and then with
different concentration of CN− ((A,E) 0 µM; (B,F) 10 µM; (C,G) 80 µM; (D,H) 160 µM) for 15 min. (A–
D) is Bright field; (E–H) is under fluorescence (λex = 385 nm, λem = 405–440 nm) blue channel (425 nm–
520 nm), scale bar: 20 µm.
Figure 6. Images of living HeLa cells: HeLa cells incubated with probe 1 (10 µM) for 1 h and then with
Figure 6.different
Imagesconcentration
of living HeLa
cells10incubated
with
1 (10
1 (A–
h and then
((A,E) HeLa
0 µM; (B,F)
µM; (C,G) 80
µM; probe
(D,H) 160
µM)µM)
for 15for
min.
of CN−cells:
−cells:
Figure
6.
Images
of(E–H)
living
HeLa
cells
with
180
(10
µM)
forchannel
1 h and
then
with
with different
concentration
of
CN
((A,E)
0 µM;
(B,F)
(C,G)
µM;
(D,H)
160(425
µM)
for 15 min.
ex
= 38510
nm,µM;
λem =probe
405–440
nm)
blue
nm–
D) is Bright
field;
isHeLa
under
fluorescence
(λincubated
− ((A,E) 0 µM; (B,F) 10 µM; (C,G) 80 µM; (D,H) 160 µM) for 15 min. (A–
ofis
CN
520
nm),concentration
scale
20 µm.
(A–D) isdifferent
Bright
field;bar:
(E–H)
under
fluorescence (λex = 385 nm, λem = 405–440 nm) blue channel
D) is Bright field; (E–H) is under fluorescence (λex = 385 nm, λem = 405–440 nm) blue channel (425 nm–
(425 nm–520 nm), scale bar: 20 µm.
520 nm), scale bar: 20 µm.
Figure 7. Images of living HeLa cells: HeLa cells incubated with different concentration of probe 1
((A,E) 0 µM; (B,F) 2.5 µM; (C,G) 5 µM; (D,H) 10 µM) for 1 h and then all cells were incubated with 80
µM CN− for 15 min. (A–D) is Bright field; (E–H) is under fluorescence (λex = 385 nm, λem = 405–440
nm), scale bar: 20 µm.
Figure 7. Images of living HeLa cells: HeLa cells incubated with different concentration of probe 1
((A,E) 0 µM; (B,F) 2.5 µM; (C,G) 5 µM; (D,H) 10 µM) for 1 h and then all cells were incubated with 80
for 15 min.
(A–D)HeLa
is Bright
under fluorescence
(λex concentration
= 385 nm, λem =of405–440
µM CN
Figure
7.− Images
of living
cells:field;
HeLa(E–H)
cells is
incubated
with different
probe 1
7.
Images
of
living
HeLa
cells:
HeLa
incubated
concentration
nm), scale
202.5
µm.µM;
((A,E)
0 µM;bar:
(B,F)
(C,G)
5 µM;
(D,H)cells
10 µM)
for 1 h andwith
then different
all cells were
incubated withof
80probe
Figure
1
− for 15
((A,E) 0µM
µM;CN(B,F)
2.5min.
µM;
(C,G)
5
µM;
(D,H)
10
µM)
for
1
h
and
then
all
cells
were
incubated
(A–D) is Bright field; (E–H) is under fluorescence (λex = 385 nm, λem = 405–440
µm.min. (A–D) is Bright field; (E–H) is under fluorescence (λex = 385 nm,
with 80nm),
µM scale
CN−bar:
for2015
λem = 405–440 nm), scale bar: 20 µm.
Sensors 2017, 17, 405
6 of 10
3. Experimental Section
3.1. Materials and Instruments
All reagents were of analytical grade and used without further purification. The absorption
and fluorescence spectra were measured on a UV-2450 (Shimadzu, Kyoto, Japan) instrument and a
F-7000 (Hitachi, Tokyo, Japan) fluorescence spectrometer. 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz)
spectra were obtained on a Bruker-300 MHz spectrometer (Bruker, Fällanden, Switzerland) using
tetramethylsilane (TMS) as an internal standard. The following abbreviations were used to denote the
signal multiplicities: s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad.
High-resolution mass spectra were obtained on a micrOTOF-Q mass spectrometer (Bruker, Bremen,
Germany). Fluorescence cell imaging was performed with a fluorescence microscope (Olympus, Tokyo,
Japan) with a 40× objective lens.
3.2. Synthesis of Probe 1
3.2.1. Synthesis of 4-bromo-N,N-di-p-tolylaniline (3)
4-Methyl-N-phenyl-N-(p-tolyl)aniline (1.00 g, 3.66 mmol) and NBS (0.68 g, 3.84 mmol) were
dissolved in CHCl3 (20 mL). The mixture was stirred for 1.5 h at room temperature. The mixture was
poured into water (50 mL) and then extracted with dichloromethane. The organic layer was washed
with plenty of brine and dried over anhydrous sodium sulfate. The solvent was evaporated by a rotary
evaporator. A white solid (1.28 g) was obtained. Yield: 99%.1 H-NMR (CDCl3 ): δ 7.26 (d, J = 9.0 Hz,
1H), 7.06 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 6.0 Hz, 1H), 2.30 (s, 3H).
3.2.2. Synthesis of 5-(4-(di-p-tolylamino)phenyl)thiophene-2-carbaldehyde (4)
Dioxane (50 mL) was placed in a 3-necked round bottom flask and was purged with nitrogen for
30 min. Under a nitrogen atmosphere, compound 3 (1.00 g, 2.84 mmol), (5-formylthiophen-2-yl)boronic
acid (1.35 g, 3.40 mmol), K2 CO3 (2.00 g, 5.68 mmol) and Pd(PPh3 )4 (164 mg, 0.14 mmol) were added.
The mixture was violently refluxed for 3.5 h. After cooling to room temperature, the solvent was
evaporated by a rotary evaporator. The residue was purified by column chromatography (silica gel,
ethyl acetate/petroleum ether = 1:20) to give the compound 4. The product was obtained as a yellow
solid (0.73 g). Yield: 67%. 1 H-NMR (DMSO-d6 ): δ 9.84 (s, 1H), 7.93 (d, J = 4.0 Hz, 1H), 7.62 (d, J = 7.6 Hz,
2H), 7.56 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 8.2 Hz, 4H), 6.98 (d, J = 8.3 Hz, 4H), 6.86 (d, J = 8.8 Hz, 2H),
2.27 (s, 6H); 13 C-NMR (DMSO-d6 ): δ 183.6, 153.2, 148.9, 143.9, 140.6, 139.5, 133.5, 130.3, 127.3, 125.3,
124.5, 123.6, 120.3, 20.5.
3.2.3. Synthesis of 2-((5-(4-(di-p-tolylamino)phenyl)thiophen-2-yl)methylene)malononitrile (1)
To a solution of compound 4 (50 mg, 0.13 mmol) and malononitrile (13 mg, 0.19 mmol) dissolved
in toluene (3 mL), and a drop of piperidine was added under a nitrogen atmosphere. The mixture was
refluxed for 3 h. After cooling to room temperature, the solvent was evaporated by a rotary evaporator.
The residue was purified by column chromatography (silica gel, ethyl acetate/petroleum ether = 1:10)
to give the title compound 1 as a red solid (47 mg). Yield: 70%. 1 H-NMR (CDCl3 ): δ 7.73 (s, 1H), 7.66
(d, J = 4.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 4.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 4H), 7.04 (d,
J = 8.3 Hz, 4H), 6.98 (d, J = 8.7 Hz, 2H), 2.35 (s, 6H); 13 C-NMR (CDCl3 ): δ 157.5, 150.3, 150.2, 143.9,
140.5, 134.2, 132.8, 130.2, 127.5, 125.6, 123.8, 122.9, 120.4, 114.6, 113.7, 74.5, 20.9; HRMS (ESI): m/z,
[M + H]+ calcd. for C28 H22 N3 S+ , 432.1529; found, 432.1516.
3.3. General Fluorescence Spectra Measurements
Figure S4 shows that the fluorescence intensity of probe 1 (5 µM) was slightly affected within
a range of pH (7.0–9.0) in the absence and presence of CN− (50 µM) in PBS/DMSO (4/6) solution.
The fluorescence responses of probe 1 were investigated in PBS/DMSO (4:6, v/v, pH = 7.4) solution.
Sensors 2017, 17, 405
7 of 10
The response time of the probe 1 towards CN− was 80 min (Figure S2). To ensure the reaction
accomplish completely, fluorescence measurements were delayed for 1.5 h after anion addition.
Probe 1 was dissolved in DMSO to afford a concentration of 5 mM stock solution and then
diluted to 5 µM with PBS/DMSO= 4/6 (v/v, pH = 7.4). Cyanide anion (10 mM) was offered by
tetrabutylammonium cyanide. Moreover, miscellaneous interference anions (as their Na+ or K+
salt, 10 mM) including S2− , HS− , SO3 2− , S2 O3 2− , S2 O8 2− , I− , Br− , Cl− , F− , NO2 − , N3 − , SO4 2− ,
SCN− , HCO3 − , CO3 2− and AcO− in deionized water were used for the sensing experiments. The
fluorescence spectrum changes caused by CN− (50 µM) and miscellaneous interference anions (50 µM)
were recorded at room temperature using a fluorescence spectrometer (λex = 370 nm, λem = 480 nm,
slits: 2.5 nm/5 nm).
3.4. Cell Culture and Confocal Fluorescence Imaging
HeLa cells were obtained from the State Key Laboratory of Biotherapy of Sichuan University.
Cells were seeded to a 12-well plates, and were routinely maintained at 37 ◦ C in a humidified 5% CO2
atmosphere using DMEM Growth medium with 10% fetal bovine serum for 24 h before use.
HeLa cells (Figure 6) were incubated with probe 1 (10 µM) for 1 h, and then washed with PBS
five times. All cells were incubated with PBS for 15 min; finally with different concentrations of CN−
were added for 1 h at 37 ◦ C: A, E with PBS; B, F with 10 µM CN− ; C, G with 80 µM CN− ; D, H with
160 µM CN− . Then cells were washed with PBS five times. Fluorescence images were monitored at
425−520 nm for blue channels and the white light channel.
Next, HeLa cells (Figure 7) were incubated with different concentrations of probe 1 for 1 h:
A, E with PBS; B, F with 2.5 µM probe 1); C, G with 5 µM probe 1; D, H with 10 µM probe 1. Then cells
were washed with PBS five times. Then all cells were incubated with 80 µM CN− for 15 min. Cell
imaging was carried out after washing cells with PBS five times.
4. Conclusions
In summary, a colorimetric fluorescent probe 1 bearing triphenylamine-thiophene and
dicyanovinyl groups has been synthesized. Probe 1 exhibited prominent selectivity and sensitivity
for detecting cyanide anion with a significant turn-on fluorescent response in PBS/DMSO (4/6, v/v,
pH = 7.4) solution. Moreover, a low detection limit (LOD, 51 nM) was achieved and it was noted
that the probe 1 has potential for detecting level of cyanide anion in drinking water according to the
requirements of the WHO (1.9 µM). In addition, good cell membrane permeability and low cytotoxicity
to HeLa cells were also observed, suggesting its promising potential in bio-imaging.
Supplementary Materials: The following are available online at www.mdpi.com/1424-8220/17/2/405/s1:
Additional spectral data for probe 1 and spectroscopic characterization of compounds 1–3.
Acknowledgments: This work was supported by the Basic Research Project of Sichuan Province (2014JY0205).
We thank the Analytical and Testing Center of Sichuan University for fluorescent measurements.
Author Contributions: Li Yang and Qing-Fei Huang conceived and designed the experiments; Zi-Hua Zheng
performed the synthetic and fluorescent experiments; Zhi-Ke Li and Lin-Jiang Song performed cell imaging;
Qi-Wei Wang contributed reagents; Li Yang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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