An unusual AIE fluorescent sensor for sequentially detecting Co2+-Hg2+-Cu2+ based on diphenylacrylonitrile Schiff-base derivative

Article abstract of DOI:10.1016/j.dyepig.2019.107590

Aggregation-induced emission (AIE)molecules with strong fluorescence in aggregated states are favourable for preparing the organic fluorescence sensor in aqueous phase. In this work, a red fluorescent sensor 5 based on AIE diphenylacrylonitrile Schiff-bas

Full text of DOI:10.1016/j.dyepig.2019.107590

Accepted Manuscript
2+
2+
2+
An unusual AIE fluorescent sensor for sequentially detecting Co -Hg -Cu based
on diphenylacrylonitrile Schiff-base derivative
Jiabin Qiu, Shengjie Jiang, Bingni Lin, Hongyu Guo, Fafu Yang
PII:
S0143-7208(19)30894-0
DOI:
https://doi.org/10.1016/j.dyepig.2019.107590
Article Number: 107590
Reference:
DYPI 107590
To appear in: Dyes and Pigments
Received Date: 22 April 2019
Revised Date: 26 May 2019
Accepted Date: 27 May 2019
Please cite this article as: Qiu J, Jiang S, Lin B, Guo H, Yang F, An unusual AIE fluorescent sensor for
2+
2+
2+
sequentially detecting Co -Hg -Cu based on diphenylacrylonitrile Schiff-base derivative, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107590.
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ACCEPTED MANUSCRIPT
An unusual AIE fluorescent sensor for sequentially detecting Co2+-Hg2+-
Cu2+ based on diphenylacrylonitrile Schiff-base derivative
Jiabin Qiu,a Shengjie Jiang,a Bingni Lin,a Hongyu Guo,*a,b and Fafu Yang*a,c
5
aCollege of Chemistry and Materials, Fujian Normal University, Fuzhou 350007, P. R. China ;Tel:
+0086-591-83465225; E-mail: yangfafu@fjnu.edu.cn
bFujian Key Laboratory of Polymer Materials, Fuzhou 350007, P. R. China
cFujian provincial Key Laboratory of Advanced Materials Oriented Chemical Engineering, Fuzhou
10 350007, P. R. China
Aggregation-induced emission (AIE) molecules with strong 30 ions was successfully applied in test paper, exhibiting good
fluorescence in aggregated states are favorable for preparing
the organic fluorescence sensor in aqueous phase. In this
practical application potential for detecting Co2+-Hg2+-Cu2+
sequentially. Sample 5 showed good bioimaging performance
on living cell. The fluorescence of living cells with sample 5
also displayed the similar change of quenching-recovering-
15 work,
a
red fluorescent sensor
5
based on AIE
diphenylacrylonitrile Schiff-base derivative was prepared in
yield of 83.7%. Sample 5 exhibited strong AIE property in 35 quenching by adding Co2+-Hg2+-Cu2+ orderly, implying the
water with red fluorescence at λex = 620 nm and the Stokes
shift was as large as 260 nm. The experiments of sensing
20 series of metallic ions suggested that sample 5 possessed the
sequentially detecting abilities for Co2+-Hg2+-Cu2+, which was
observed firstly for organic fluorescence sensor. The
fluorescence of sample 5 was quenched rapidly in presence of
Co2+, then recovered by adding Hg2+ and quenched once more
25 by further adding Cu2+. The detection limits for Co2+, Hg2+
and Cu2+ were 0.0937, 0.0221, and 0.0988 µM, respectively.
The sensor mechanism was confirmed by fluorescence
titration, 1H NMR and MS spectra, indicating the 2:1
stoichiometric ratios of sample 5 with ions. The sensor for
good ions-detecting prospect in living body environment.
Keywords: Diphenylacrylonitrile; Fluorescence; Sensor; Co2+;
Hg2+; Cu2+
1. Introduction
40 Contamination of heavy metals seriously threatens to human’s
health and causes series of diseases usually [1,2]. For
instance, copper induces Menkes syndrome, cobalt leads to
radiation skin damages, and mercury disturbs the central
neural system [3-5]. Thus, it is very important to develop the
45 timely and efficient detection method for heavy metals ions
among all kinds of complicated environment such as mine

wastewater, industrial sewage, even living organisms.
metallic ions has the advantage of higher efficiency, cheaper
cost and more simplified operation in comparison with the
sensor for one metallic ion. Up to now, few examples
ACCEPTED MANUSCRIPT
Traditional detection methods including atomic absorption
spectroscopy (AAS), atomic fluorescence spectrometry
(AFS), X-ray fluorescence spectrographic (XFS) and 35 involved the sensing abilities for two kinds of metallic heavy
5
inductively coupled plasma mass spectrometry (ICP-MS)
[6,7], are successfully used in heavy metals detection.
However, although those testing instruments are sensitive and
accurate in general, they were restricted for its huge
equipment, complex and professional operation.
ions, such as for Hg2+ and Ag+, Zn2+ and Cd2+, or Fe3+ and
Hg2+ [20, 43, 44]. In this paper, we wish to report an unusual
AIE fluorescent sensor for three kinds of heavy ions for the
first time. This sensor was a simple diphenylacrylonitrile
40 Schiff-base derivative, exhibiting the sequentially detecting
abilities for Co2+ with fluorescence quenching, Hg2+ with
fluorescence enhancement, and Cu2+ with fluorescence
quenching again. Moreover, it was successfully applied in test
paper and bioimaging.
10
Recently, the fluorescent sensors attracted much attention
in detecting ions due to their obvious advantages of high
sensitivity, low cost, and simple operation [8-13]. Many
traditional dye molecules with excellent photoluminescence,
such as BODIPY, fluorescein dyes, rhodamine dyes and
45
2. Experimental
15 cyanine dyes were modified as excellent fluorescent probes
for sensing anionic ions, cation ions, biological molecules, etc
[14-21]. These fluorescent probes have been usually studied
in organic solvents or organic/aqueous mixtures solutions,
limiting their practical application due to the strong
20 aggregation-caused quenching (ACQ) effect in aqueous
solution. In 2001, Tang’s group reported the aggregation
induced emission (AIE) phenomenon for the first time [22].
AIE molecules can effectively eliminate the ACQ effect,
resulting in the widely application in numerous fields, such as
25 luminous liquid crystal [23-26], organic light-emitting diodes
(OLED) [27-29], circularly polarized photoluminescence [30-
32], ion detection and biomolecular recognition in water [33-
42].
2.1 General
All chemical reagents were obtained from J&K Chemicals
50 and were used directly. TLC analysis was carried out on pre-
coated glass plates. Silica gel (200-300 mesh) was used for
the purification of rapid column chromatography. NMR
spectra were recorded on a Bruker-ARX 400 instrument at 26
oC. MS spectra were examined on Bruker mass spectrometer.
55 UV-Vis spectra were measured on Varian UV-Vis
spectrometer. Fluorescence spectra were investigated in a
conventional quartz cell (10×10×45 nm) on a Hitachi F-4500
o
spectrometer at 26 C. The fluorescence absolute ΦF values
were obtained on FLS920 Fluorescence spectrometer with a
60 6-inch integrating sphere. The MCF-7 cancer cells were
supplied by the school of Pharmacy, Fujian Medical
University.
Usually, both the traditional dye probes and AIE probes
30 exhibited the selectively sensing abilities for one kind of
metallic ions. Obviously, the selective sensor for multiple

2.2. Synthetic procedure for compound 3
1H, C=CH). 11.92 (d, J = 8.0 Hz, 1H, ArH), 8.12 (bs, 1H,
OH). HR-MS (ESI) (C16H10N2O4) Anal. calcd: 294.0641.
Found: 294.0632.
ACCEPTED MANUSCRIPT
Under the protection of nitrogen, 4-hydroxybenzaldehyde
(2.43 g, 15 mmol) and 4-nitrophenylacetonitrile (1.83 g, 15
mmol) were dissolved in 100 mL of dry dichloromethane with 35 2.4 Synthetic procedure for compound
5
5
1.5 mL of piperidine in the reaction solution. The mixture was
stirred at room temperature for 5 h. The colour of the solution
Under the protection of nitrogen, compound 4 (0.59 g, 2
mmol) with 2-aminopyridine (0.19 g, 2 mmol) were dissolved
in 20 mL of MeOH with five drops of glacial acetic acid as
catalyst. The mixture was stirred and refluxed overnight and
changed to
a
burgundy, accompanying with brown
precipitation. When all of the starting materials disappeared
by the TLC detection, the reaction was stopped and 40 the dark red precipitate appeared. After evaporating the
10 neutralized with 10% of dilute hydrochloric acid. The organic
layer was partitioned and the solution was distilled. The
residue was recrystallized in CHCl3-MeOH and compound 3
solvent under vacuum distillation, the residue was purified
with recrystallization in MeOH. The target compound 5 was
1
obtained as blood-red solid in yield of 83.7%. H NMR (400
1
was obtained as yellowish solid in yield of 87.6%. H NMR
MHz, DMSO-d6), δppm: 10.34 (s, 1H, N=CH), 8.36 (s, 1H,
(400 MHz, CDCl3): δppm: 13.03 (d, J = 12.0 Hz, 2H, ArH), 45 ArH), 8.33 (d, J = 8.0 Hz, 2H, ArH), 8.25 (s, 1H, C=CH),
15 12.83 (s, 1H, C=CH), 12.68 (d, J = 12.0 Hz, 2H, ArH), 12.63
(d, J = 12.0 Hz, 2H, ArH), 11.63 (d, J = 12.0 Hz, 2H, ArH),
9.45 (bs, 1H, OH). MALDI-TOF-MS (C15H10N2O3) calcd for
m/z = 266.0691, found: m/z = 266.0664 (M+).
8.18 (d, J = 8.0 Hz, 1H, ArH), 8.01 (d, J = 8.0 Hz, 2H, ArH),
7.87 (bs, 1H, ArH), 7.34-7.38 (m, 1H, ArH), 7.15(d, J = 12.0
Hz, 1H, ArH), 6.43-6.48 (m, 2H, ArH), 5.89 (bs, 1H, OH). 13
C
NMR (100 MHz, DMSO-d6), δppm: 190.14, 164.28, 159.93,
50 149.54, 147.32, 145.63, 140.88, 137.69, 137.09, 131.35,
126.99, 124.73, 123.32, 120.27, 118.95, 117.86, 112.32,
108.62, 105.68. HR-MS (ESI) (C21H14N4O3) [MH]+: Anal.
calcd: 371.1146(MH+). Found: 371.1189(MH+).
2.3 Synthetic procedure for compound 4
20 In nitrogen atmosphere, compound 3 (2.66 g, 10 mmol) and
urotropin (2.80 g, 20 mmol) were dispersed in 30 mL of
trifluoroacetic acid. The mixture was stirred and refluxed for
12 h. TLC detection suggested the dissapearance of materials.
2.5 MTT Assay
Then trifluoroacetic acid in the reaction system was 55 Methylthiazolyldiphenyl-tetrazolium (MTT) trials were done
25 evaporating by vacuum distillation, and the residues were
further separated and purified by chromatographic silica gel
column (eluent: dichlormethane/petroleum ether = 3:1).
Compound 4 was collected in yield of 46.3% as orange solid.
to determine the toxicity for MCF-7 cancer cells. The
o
Inoculated MCF-7 cancer cells were cultivated at 37 C and
5% CO2 for 24 hours, then 1.0×10-5 M of sample 5 was
tracked in the cells after incubating for 24 h. The fostered
1H NMR (400 MHz, DMSO-d6), δppm: 13.08 (s, 1H, ArH), 60 cells were rinsed by PBS buffer, and continue cultivating for 3
30 13.06 (d, J = 8.0 Hz, 2H, ArH), 13.00 (s, 1H, CHO), 12.93 (d,
J = 8.0 Hz, 1H, ArH), 12.73 (d, J = 8.0 Hz, 2H, ArH), 12.51 (s,
hours, in 0.5 mg/mL MTT-PBS buffer. Further, 100 µL of

DMSO was added into dissolve the generated Formazan
condensation
of
4-hydroxybenzaldehyde
and
4-
ACCEPTED MANUSCRIPT
crystals, and detected the absorption intensity at 490 nm.
2.6 The experiment of living cell imaging
30 nitrophenylacetonitrile in dry dichloromethane, with equal
molar ratio of piperidine as catalyst. Compound 3 was further
converted to compound 4 in yield of 46.3% via the classic
Duff-reaction under the refluxing reaction system of
hexamethylenetetramine and trifluoroacetic acid. Finally, the
Compound 5 (3.0 mg) was dissolved in 1 mL of DMSO and
then diluted with PBS buffer (pH = 7.4) to concentration of
1.0×10-5 M for imaging test. MCF-7 cancer cells were
5
cultivated at the above identical circumstance of MTT trials 35 target compound 5 was synthesized by the Schiff-base
for 24 h, and then being tinted by 1.0×10-5 M of sample 5 with
condensation of compound 4 and 2-amino pyridine. The yield
was as high as 83.7% after recrystallization. The structure of
target compound 5 was confirmed by 1H NMR, 13C NMR, and
HR-MS spectral analyses (see the ESI). The molecular ion
24 h breeding. After rinsing with PBS-buffer, dyed cells were
10 cultivated in solution of metallic ions (1.0×10-5 M) for 1 hour
o
at 37 C, respectively. The cells were imaged by a confocal
laser scanning microscope (CLSM, Zeiss LSM 710, Jena, 40 peak (MH)+ at 371.1189 in the HR-MS spectra agreed well
Germany).
with the accurate value of the molecular weight of compound
1
5. The signals of the H NMR spectra and 13C NMR spectra
15 3. Results and discussion
were also in accordance with the structure.
3.1. Synthesis and characterization
3.2. AIE properties
NO2
NO2
NO2
100.0k
N
0%
10%
20%
30%
40%
50%
NH2
NO2
OH
N
N
CN
N
N
piperidine
DCM
80.0k
+
CN
CN
MeOH, reflux
TFA, refluxing
OHC
CHO
CN
N
OH
60%
60.0k
2
1
OH
70%
80%
85%
90%
95%
99%
OH
N
5
4
3
Scheme 1 The synthetic route for target compound 5
40.0k
20.0k
0.0
20 In order to sense heavy ions effectively, the target compound
was designed by introducing the pyridine unit, Schiff-base
group and phenolic group into diphenylacrylonitrile skeleton
to produce the multiple N and O binding sites for metallic
ions. The nitrophenylacetonitrile was used for increasing the
25 molecular polarity, which was in favour of the long-
wavelength fluorescence. The synthetic route was illustrated
in Scheme 1. Briefly, the diphenylacrylonitrile derivative 3
was prepared in yield of 87.6% at room temperature by
500
550
600
650
700
750
800
Emission Wavelength (nm)
45
Figure 1. Fluorescence spectra of sample 5 (1 µM) in THF
and THF-H2O mixtures with different water fractions, λex
=
360 nm.
50 The AIE properties of compound 5 were studied by UV and
photoluminescence spectra in THF-H2O mixtures. Figure 1
illustrated the corresponding fluorescent spectra in THF-H2O

mixtures and the plot of fluorescence intensities versus the
low background noise and high signal-to-noise ratio of
biological resources [45].
ACCEPTED MANUSCRIPT
water fractions was displayed in Figure 2. It can be seen that
sample 5 exhibited certain fluorescence intensity in pure THF,
which decreased rapidly with the increase of H2O fractions
and kept at the low levels when fwater < 80%. As fwater > 80%,
the fluorescence intensities enhanced dramatically. The
fluorescence intensity at fwater = 99% was 30 times higher than
that in 70% of water fraction. The particle size distribution of
sample 5 (1 µM) in solution of THF-H2O (1:99) was also
100.0k
80.0k
60.0k
40.0k
20.0k
0.0
5
10 evaluated by dynamic light scattering (DLS). The results
suggested that the average size distribution of sample 5 were
100 nm (Figure S6). The outstanding fluorescence in solid
state was also observed for sample 5 as shown in Figure S7.
Moreover, the fluorescence quantum yields of sample 5 in
15 pure THF solution, THF-H2O with 99% of water fractions and
solid state were determined as 1.1%, 24.6% and 26.8%,
respectively. These results certainly implied the strong AIE
effect for sample 5 in aggregated state. On the other hand, the
maximum fluorescence wavelength showed some red shift
20 with the increase of water fractions. These phenomena could
be explained by the TICT effect due to the electron-donating
abilities of the hydroxyl group and the electron-withdrawing
abilities of the cyano and nitro group. Based on this strong
molecular polarity, sample 5 exhibited the rose-bengal
25 luminescence with emission wavelength at 620 nm and the
Stokes shift was as large as 260 nm (as shown in Figure S8),
which was seldom observed for diphenylacrylonitrile
derivative. The strong long-wavelength fluorescence with
large Stokes shift was favourable for the detection due to its
30 advantages of deep tissue infiltration, minimal self-absorption,
0.0
0.2
0.4
0.6
0.8
1.0
Water fraction (vol%)
Figure 2. Plot of fluorescence intensities versus the water
fractions of THF-H2O mixtures. Insert: The fluorescence
35 images of sample 5 (1 µM) in THF-H2O with water fractions
of 0%, 40%, 85% and 99%, respectively.
3.3 The sensor for metallic ions
The sensor abilities of compound 5 for metallic ions were
40 investigated by measuring the fluorescence change of sample
5 with all kinds of cations. As shown in Figure 3, the
fluorescences of sample 5 were little influenced by Ag+, Al3+,
Ba2+, Bi3+, Fe3+, Hg2+, K+, Mg2+, Na+, Ni2+, Pb2+, Zn2+, Cr2+,
Ca2+, Li+ and Cd2+ with the small fluctuation of fluorescence
45 intensity. The fluorescence wavelength of sample 5 with Ag+
and Hg2+ exhibited a little blue shift, which could not be
distinguished by naked eye. However, it could be seen that the
fluorescence of sample 5 after binding with Co2+ and Cu2+
exhibited strong fluorescence quenching. The fluorescence
50 was almost invisible (inserted picture of Figure 5). These
results suggested that sample 2 was an excellent sensor for
Co2+ and Cu2+.

ACCEPTED MANUSCRIPT
250.0k
+
0
(A)
Ag
1*10-6
2*10-6
3*10-6
4*10-6
5*10-6
6*10-6
7*10-6
8*10-6
9*10-6
1*10-5
3+
Al
80000
60000
40000
20000
0
Hg2+
Ag+
2+
Ba
200.0k
2+
25
Bi
2+
Co
2+
Cu
3+
150.0k
Fe
2+
Hg
+
K
2+
Mg
100.0k
+
Na
2+
Ni
2+
Pb
50.0k
2+
Zn
3+
Cr
Co2+
Cu2+
2+
Ca
+
0.0
Li
2+
500
600
700
800
Cd
None
Emission Wavelength(nm)
400
500
600
700
800
30
250.0k
200.0k
150.0k
100.0k
50.0k
0.0
Emission wavelength (nm)
(B)
250.0k
Equation y = a +
Adj. R-Squ 0.9801
200.0k
150.0k
100.0k
50.0k
0.0
Figure 3. Fluorescence spectra of sample 5 (0.1 µM) with
different metal cations (0.1 µM) in THF-H2O mixtures with
99% of water fractions. λex= 360 nm.
Value
Standard E
B
B
Intercep 209846.58 7964.2785
Slope -41380.217 2630.5146
0
1
2
3
4
5
35
5
3.4 Titration of Co2+ and Cu2+
0
2
4
6
8
10
Emission Wavelength(nm)
250.0k
(A)
0
1*10-6
2*10-6
Figure 5. (A) Fluorescence spectra of sample 5 (10 µM) with
different concentrations of Cu2+ in THF-H2O (1:99), λex= 360
nm. (B) The fluorescence intensities of probe titration with
200.0k
3*10-6
4*10-6
5*10-6
6*10-6
150.0k
7*10-6
8*10-6
9*10-6
1*10-5
100.0k
40 Cu2+.
10
50.0k
0.0
500
600
700
800
The further investigation of the sensing properties of sample 5
Emission Wavelength(nm)
for Co2+ and Cu2+ were carried out by the fluorescence
titration experiments. As shown in Figure 4(A) and 5(A), with
45 the increase of the concentration of Co2+ or Cu2+, the
intensities of the emission of sample 5 decreased rapidly and
the fluorescence was almost quenched completely when 0.5
equiv of Co2+ or Cu2+ was added in. These results might
indicate the 2:1 of stoichiometric ratio for sample 5 with Co2+
50 or Cu2+, which was further confirmed by the following work.
Based on the changes of fluorescence spectra, the
corresponding plot of fluorescence intensities versus
concentrations of Co2+ or Cu2+ was illustrated in Figure 4(B)
(B)
Equation
200.0k
y= a+ b*
200.0k
150.0k
100.0k
50.0k
0.0
Adj. R-Squar 0.98277
Value
Intercept 194257.3613 7056.43796
Slope -39431.4075 2330.66475
StandardErro
B
B
150.0k
100.0k
50.0k
0.0
15
0
1
2
3
4
5
0
2
4
6
8
10
[Co2+] (
µM)
Figure 4. (A) Fluorescence spectra of sample 5 (10 µM) with
20 different concentrations of Co2+ in THF-H2O (1:99), λex = 360
nm. (B) The fluorescence intensities of probe titration with
Co2+.

and 5(B), respectively. It can be seen a good linear
suggested the system of sample 5 + Co2+ possessed the
ACCEPTED MANUSCRIPT
relationship for the systems with 0.0 to 5.0 µM metallic ions. 25 selectively sensing abilities for Hg2+. As to the system of
According to the calculation formula DL = K × Sb1/S (where
K = 2 or 3, in this case the value = 2, Sb1 is the standard
deviation of the blank solution, S is the value of the slope of
the regression line), the detection limit can be calculated as
DL = 0.0937 µM for Co2+ and 0.0894 µM for Cu2+,
respectively.
sample 5 + Cu2+, the fluorescence quenching could not be
influenced by adding other metallic ions as shown in Figure 7.
5
6k
Cu2++Ag+
Cu2++Al3+
Cu2++Ba2+
5k
Cu2++Bi2+
5
Cu2++Co2+
Cu2+
4k
3k
2k
1k
0
Cu2++Fe3+
Cu2++Hg2+
Cu2++K+
Cu2++Mg2+
Cu2++Na+
Cu2++Ni2+
Cu2++Pb2+
None
Cu2++Zn2+
Cu2++Cr3+
Cu2++Ca2+
Cu2++Li+
5+Cu2+
10 3.5 Interference experiments
Cu2++Cd2+
500
600
700
800
Co2++Ag+
Co2++Al3+
Co2++Ba2+
Co2++Bi2+
Co2+
Co2++Cu2+
Co2++Fe3+
Co2++Hg2+
Co2++K+
Co2++Mg2+
Co2++Na+
Co2++Ni2+
Co2++Pb2+
None
100.0k
Wavelength (nm)
5
Figure 7. Fluorescence spectra of sample 5 (1 µM), sample 5
and Cu2+(1 µM) or Cu2+(1 µM) along with other species (1 µM)
30 in THF-H2O (1:99), λex = 360 nm.
5+Co2++Hg2+
80.0k
60.0k
40.0k
5+Co2+
Co2++Zn3+
Co2++Cr3+
Co2++Ca2+
Co2++Li+
20.0k
3.6 The sequentially sensor for Co2+-Hg2+-Cu2+
Co2++Cd2+
0.0
500
600
700
800
As the interference experiments suggested the fluorescence of
sample 5 + Co2+ could be lightened by adding Hg2+ but the
35 fluorescence of sample 5 + Cu2+ had no the same phenomenon,
thus, the unusual fluorescent sensor for sequentially detecting
Co2+-Hg2+-Cu2+ could be constructed based on these results.
The sequentially detecting procedure for detecting Co2+-Hg2+-
Cu2+ was illustrated in Figure 8. The red fluorescence was
40 firstly quenched in presence of Co2+ and then lightened again
by adding Hg2+. After Cu2+ was further added in, the
fluorescence was quenched once more. Based on these
phenomena, the sequentially sensor for Co2+-Hg2+-Cu2+ were
studied by the fluorescence titration experiments.
Wavelength (nm)
Figure 6. Fluorescence spectra of sample 5 (1 µM), sample 5
and Co2+(1 µM) or Co2+(1 µM) along with other species (1 µM)
in THF-H2O (1:99), λex = 360 nm.
15 To study the interference of other cations, the sensing
selectivity of sample 5 for Co2+ and Cu2+ were investigated by
competition experiments with other metal ions. The results
were exhibited in Figure 6 and 7. It can be seen that, sample 5
and Co2+ with other metallic ions maintained fluorescence
20 quenching or weak fluorescence except Hg2+. The
fluorescence almost completely restored for sample 5 with
Co2+ and Hg2+. The maximum wavelength displayed a little
blue shift as in previous sensing experiment. This result
45

suggest 2:1 of stoichiometric ratio for sample 5 with Hg2+.
ACCEPTED MANUSCRIPT
Figure 9(B) illustrated the corresponding plot of fluorescence
30 intensities versus concentrations of Hg2+. A good linear
relationship for the systems with 0.0-0.5 µM of Hg2+ could be
observed. The detection limit could be calculated as 0.0221
µM for Hg2+ according to the previously mentioned
calculation formula DL = K × Sb1/S.
Figure 8. The changes of fluorescent photos of sample 5 for
sequentially detecting Co2+-Hg2+-Cu2+ (under UV light, 365
nm)
35
30.0k
(A)
0
5+Co2++Hg2+
0
25.0k
20.0k
15.0k
10.0k
5.0k
5
10
15
1*10-7
1*10-7
2*10-7
3*10-7
4*10-7
5*10-7
6*10-7
7*10-7
8*10-7
9*10-7
1*10-6
5+Co2++Hg2+
25.0k
2*10-7
Cu2+
3*10-7
4*10-7
20.0k
5*10-7
5+Co2++Hg2++Cu2+
6*10-7
15.0k
Hg2+
7*10-7
10.0k
5.0k
0.0
8*10-7
9*10-7
1*10-6
5+Co2+
40
500
600
700
800
Wavelength (nm)
0.0
(B)
Equation
y
=
a
+
b*x
500
600
Wavelength (nm)
700
800
25.0k
20.0k
15.0k
10.0k
5.0k
25.0k
Adj. R-Square
0.98996
Value
25133.04762
-4592.28571
Standard Error
625.56707
206.618
B
B
Intercept
Slope
20.0k
15.0k
10.0k
5.0k
25.0k
20.0k
15.0k
10.0k
5.0k
0.0
0
1
2
3
4
5
45
25.0k
20.0k
15.0k
10.0k
5.0k
0.0
0
2
4
6
8
10
[Cu2+] (
µM)
Equation
y
=
a
+
b*x
Adj. R-Square
0.99177
Value
3065.52381
4129.85714
Standard Error
B
B
Intercept
Slope
508.83632
168.0631
Figure 10. (A) Fluorescence spectra of sample 5 (1 µM) +
0.0
0
1
2
3
4
5
0.0
50 Co2+ (1 µM) + Hg2+ (1 µM) with different concentrations of
Cu2+ in THF-H2O (1:99), λex= 360 nm. (B) The fluorescence
intensities of probe titration with Cu2+.
0
2
4
6
8
10
[Hg2+] (10-7 M)
20 Figure 9. (A) Fluorescence spectra of sample 5 (1 µM) + Co2+
(1 µM) with different concentrations of Hg2+ in THF-H2O
(1:99), λex= 360 nm. (B) The fluorescence intensities of probe
titration with Hg2+.
Furthermore, the mixture systems of sample 5, Co2+ and
55 Hg2+ were treated with different concentrations of Cu2+. The
fluorescence was quenched gradually once more with the
increase of concentration of Cu2+ as shown in Figure 10. The
turning point for fluorescence change also appeared at 0.5 µM
As shown in Figure 9(A), with the increase of the
25 concentration of Hg2+, the intensities of the emission of the
system of sample 5 and Co2+ enhanced rapidly and attained
the maximum value at about 0.5 µM of Hg2+, which might

of Cu2+, indicating the 2:1 of stoichiometric ratio for sample 5
obvious shifts to low field, indicating the Cu2+ was binded in
the cavity composed of N and O functional groups as inserted
in Figure 12. Furthermore, the binding constant Ka was
ACCEPTED MANUSCRIPT
with Cu2+ again. According to calculation formula DL = K ×
Sb1/S, the detection limit was calculated as 0.0938 µM for
Cu2+. This value was a little bigger than that of the previous 35 calculated by Benesi-Hildebrand equation (FigureS15-S17).
5
system of sample 5 with Cu2+ (0.0894 µM). The difference
might be attributed to the influence of Co2+ and Hg2+ in these
systems. These above fluorescence titration results confirmed
that sample 5 possessed the sensing abilities for sequentially
The Ka values exhibited the order of Co2+ (4.06×1011) < Hg2+
(2.23×1013) < Cu2+ (2.87×1013), which agreed with the
changes of fluorescence of sample 5 in the presence of Co2+,
Hg2+ and Cu2+. Thus, the whole sensing mechanism could be
detecting Co2+-Hg2+-Cu2+, which was reported for the first 40 elucidated as Figure 13.
10 time. In addition, the influences of pH on fluorescence
intensities of sample 5 with ions were investigated as shown
in Figure S9, exhibiting a reasonable stability of fluorescence
intensity between pH = 6 and 10.
15 3.7 The sensing mechanism
The stoichiometric ratios of sample 5 for Co2+, Hg2+ and Cu2+
were investigated by the Job’s plots as shown in Figure S10-
12. It can be seen that the break points for fluorescence
intensities were at 0.33, suggesting the 2:1 binding
20 stoichiometric ratios of sample 5 for these metallic ions.
Moreover, the MS spectrum of sample 5 with excess Cu2+,
Co2+ and Hg2+ were examined as shown in Figure 11, Figure
S13 and S14, respectively. The strong peaks at m/z of 805.796,
400.380 and 941.629 were in accordance with the molecular
25 weight of 2M+Cu2+, (2M+Co2+)/2 and 2M+Hg2+ certainly
supporting the 2:1 binding stoichiometric ratio again.
Figure 11. The mass spectrum of sample 5 with excess Cu2+
1
The contrast H NMR spectra of sample 5 with Cu2+were
investigated to study the binding behaviour. As shown in
Figure 12, the proton signals displayed obvious changes after
30 binding Cu2+. Especially, the protons of OH groups
disappeared and the protons of CH=N groups displayed
Figure 12. The contrast 1H NMR spectra of sample 5 and
sample 5 with Cu2+
45

25 the fluorescence quenched once more. These color
ACCEPTED MANUSCRIPT
NO2
NO2
NO2
changes of tested paper were in accordance with the
fluorescence studies in solution, indicating the good
CN
CN
CN
NO2
application prospect of sample
Cu2+ sequentially.
5
for sensing Co2+-Hg2+-
Cu2+
-Hg2+
N
O
N
O
Hg2+
-Co2+
N
O
Co2+
-2H+
CN
N
N
N
Cu
Co
2
Hg
N
N
N
O
N
O
N
O
N
N
OH
N
NC
NC
NC
NO2
NO2
NO2
(Ka = 4.06×1011
)
(Ka = 2.87×1013
)
(Ka = 2.23×1013
)
Figure 13. The sequentially sensing mechanism of sample 5
for Co2+, Hg2+ and Cu2+
3.8 Application in test paper
30 Figure 14. Fluorescence photographs of the test papers
containing different species under UV light (365 nm). A =
None, B = Ag+, C = Al3+, D = Ba2+, E = Na+, F = Pb2+, G =
Ca2+, H = Cd2+, I = Zn2+, J = K+, K = Fe3+, L = Ni2+, M = Li+,
N = Hg2+,O = Bi3+, P = Co2+, Q = Cu2+, R = Mg2+ and S = Cr2+,
35 respectively.
5
Test paper is the convenient method for the application of ion
detection due to its portable and responsive peculiarities.
Therefore, the test paper of sample 5 was obtained by
tailoring filter paper into strips and immersed in 1 mM of
sample 5 THF solution for 1 h. After drying in air, all test
10 papers were fixed in the circular eyelet template. Furthermore,
three drops of 0.1 mM solutions (H2O/THF = 99:1),
, ,
containing none, Ag+ Al3+, Ba2+, Na+, Pb2+, Ca2+, Cd2+ Zn2+,
K+, Fe3+, Ni2+, Li+ Hg2+, Bi3+, Co2+, Cu2+, Mg2+ and Cr2+
,
,
Figure 15. Photographs of the test papers, containing multiple
species under UV light (365 nm). A = None, B = Co2+, C =
Co2+ and other tested ions except Hg2+, D = Co2+ and Hg2+, E
= Co2+, Hg2+ and Cu2+.
respectively, were added to the test papers. As shown in
15 Figure 14, there was no color change for most of ions
except for Co2+ and Cu2+, indicating the sensing abilities
for Co2+ and Cu2+. Moreover, the sequentially sensor of
test paper for Co2+-Hg2+-Cu2+ was studied. As shown in
Figure 15, after adding Co2+ on test paper, the strong red
20 fluorescence of tested paper (A) turned into no
luminescence (B). Further dropping other metallic ions
on test paper, the test paper still maintained no
luminescence (C) except Hg2+(D) exhibiting strong red
fluorescence again. Further dropping Cu2+ on tested paper,
40
3.9 Application in living cell imaging
In recent years, the living cell imaging of fluorescent
probe exhibited the broad application prospects. A study
of sample
5 was carried out by confocal laser scanning
45 microscopy (CLSM) to explore imaging performance in
biologic cells. According to the reported method of
metabolic activity with MTT assay, sample
5 showed low

29.3 µM (Figure S19). After adding ions of Co2+-Hg2+-
ACCEPTED MANUSCRIPT
Cu2+ in solution, the alive MCF-7 cell still attained 73%,
69% and 65%, respectively (Figure S20). Afterwards,
20 sample
5 was tracked in MCF-7 cells by an hour of
incubation. A bright orange-red fluorescence can be
observed in the cell as shown in Figure 16. With the
addition of Co2+, the fluorescence became dim, further
became bright again by adding Hg2+ and quenched finally
25 by adding Cu2+. These phenomena suggested that sample
5
possessed the good living cell imaging performance and
could sense Co2+-Hg2+-Cu2+ sequentially in living system.
4. Conclusion
30 In summary, this work synthesized a red fluorescent sensor 5
based on AIE diphenylacrylonitrile with pyridine Schiff-base
structure. Sample 5 exhibited good AIE property with strong
red fluorescence at λex = 620 nm and the Stokes shift was as
large as 260 nm. The experiments of sensing series of metallic
35 ions suggested that the fluorescence of sample 5 was
quenched rapidly by adding Co2+ and Cu2+. Moreover, the
quenched fluorescence of sample 5 with Co2+ could be
recovered by further adding Hg2+, and then quenched again by
adding Cu2+. These results suggested the sequentially
40 detecting abilities for Co2+-Hg2+-Cu2+ which was observed for
the first time. The detection limits for Co2+-Hg2+-Cu2+ were
0.0937, 0.0221, and 0.0938 µM, respectively. The sensor
mechanism of 2:1 stoichiometric ratios was proposed and
confirmed by fluorescence titration, 1H NMR and MS spectra.
45 The test paper of sample 5 was prepared and applied
successfully to the practical detection for Co2+-Hg2+-Cu2+
sequentially. The experiment of living cell imaging suggested
Figure 16. Confocal fluorescence images of MCF-7 cells
(1.0×10-5 M)
before and after incubated with sample
5
and/or ions. (A)-(C) sample
- MCF-7 cells- Co2+; (G)-(I) sample
cells- Co2+-Hg2+; (J)-(L) sample - MCF-7 cells- Co2+-
5-MCF-7 cells; (D)-(F)
sample
5
5- MCF-7
5
5
Hg2+-Cu2+. Left images were bright field images, middle
images were fluorescence images, and right images were
the merged images of fluorescence and bright field. (λex =
405 nm). The same scale bar for all images (as shown for
10 image J).
biotoxicity with above 80% of the alive MCF-7 cell at
o
concentration of 1.0×10-5 M for 24 h at 37 C (Figure
S18). The influence of concentrations on the cytotoxicity
15 was studied, suggesting that the half-maximal inhibitory
concentration (IC50) of sample
5 for MCF-7 cell was

the good bioimaging performance of sample 5. The
[6] Salazar MT, Smith TK, Harris A. High-performance
liquid chromatographic method for determination of
biogenic amines in feedstuffs, complete feeds, and animal
tissues. J Agric Food Chem 2000;48:1708-1712.
ACCEPTED MANUSCRIPT
fluorescence of living cells with sample 5 was also quenched
by Co2+, restored by further adding Hg2+, and quenched finally
in presence of Cu2+, implying the good application prospect in
detecting Co2+-Hg2+-Cu2+ for living body environment.
5
35 [7] Zhang Y, Peng C, Ma X, Che Y, Zhao J. Fluorescent and
photoconductive nanoribbons as a dual-mode sensor for
selective discrimination of alkyl amines versus aromatic
amines. Chem Commun 2015;51:15004-15007.
Acknowledgment. Financial support from the National
Natural Science Foundation of China (No: 21406036), Fujian
Natural Science Foundation of China (No. 2017J01571),
[8] Feuster EK, Glass TE, Detection of amines and
10 Fujian Science and Technology Project (No. 2019N0010),
Fuzhou science and technology project (2018-G-44), and
Undergraduate innovation program of FJNU (2019) were
greatly acknowledged.
40
45
50
55
60
unprotected amino acids in aqueous conditions by
formation of highly fluorescent iminium ions. J Am Chem
Soc 2003;125:16174-16175.
[9] Takagai Y, Nojiri Y, Takase T, Hinze WL, Butsugan M,
Igarashi S. "Turn-on'' fluorescent polymeric microparticle
sensors for the determination of ammonia and amines in the
vapor state. Analyst 2010;135:1417-1425.
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10

ACCEPTED MANUSCRIPT
Novel red AIE fluorescence sensor was prepared with Stokes shift of 260 nm.
The sequentially detecting abilities for Co2+-Hg2+-Cu2+ was firstly observed.
It showed good bioimaging performance on living cell for sensing Co2+-Hg2+-Cu2+.
The detection limits for Co2+, Hg2+ and Cu2+ were 0.0937, 0.0221, and 0.0988 µM.
The sensor mechanism was confirmed as 2:1 stoichiometric ratio.