Dual-functional chemical sensor for sensitive detection and bioimaging of Zn2+ and Pb2+ based on a water-soluble polymer

Article abstract of DOI:10.1016/j.orgel.2020.105711

Free lead and zinc ions are toxic to living cells and environment, we develop a multi-responsive fluorescence probe based on water-soluble double hydrophilic block copolymer. The polymer sensor with Schiff moiety can sensitively, selectively and simultaneously fluorescence probe Zn²⁺ (green) and Pb²⁺ (blue) through the different fluorescence color. The linear concentration ranges for Pd²⁺ and Zn²⁺ were 0.8–10.0 μM and 0–10.0 μM with the detection limits of 0.7 μM and 0.2 μM, respectively. The probe responding to Na⁺, Mn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺, Cd²⁺, Al³⁺, Cr³⁺, Co²⁺, Ni²⁺, Ag⁺, Ca²⁺, K⁺, Mg²⁺, Ba²⁺, Hg²⁺, and Pb²⁺ displays almost no significant interference with the assay process. The sensing mechanism of the PSAM probe to Pb²⁺ and Zn²⁺ was investigated by Job's plot experiment, ¹H NMR analysis and density functional theory (DFT), the results show that the nitrogen atom of CH[dbnd]N and oxygen atom of OH may recognize with ions and the Job's plots for the PSAM complexes demonstrate 1:1 complexation, respectively. To achieve its application, the chemosensor is successfully applied to monitor Pd²⁺and Zn²⁺ in tap and lake water samples, its ability to monitor free Pd²⁺ and Zn²⁺ ions in living cells or in vivo is also validated by the intracellular imaging.

Full text of DOI:10.1016/j.orgel.2020.105711

Organic Electronics 82 (2020) 105711
Contents lists available at ScienceDirect
Organic Electronics
journal homepage: http://www.elsevier.com/locate/orgel
Dual-functional chemical sensor for sensitive detection and bioimaging of
Zn2þ and Pb2þ based on a water-soluble polymer
,
,
,*
Gang Weia, Gang Zhaoa, Naibo Linb **, Shanyi Guangc ***, Hongyao Xua
a State Key Laboratory for Modification of Chemical Fibers and Polymers Materials, College of Materials Sciences and Engineering, Donghua University, Shanghai,
201620, China
b Research Institution for Biomimetics and Soft Matter, Fujian Key Provincial Laboratory for Soft Functional Materials Research, College of Materials, Xiamen University,
422 Siming Nan Road, Xiamen, 361005, PR China
c School of Chemistry, and Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Free lead and zinc ions are toxic to living cells and environment, we develop a multi-responsive fluorescence
probe based on water-soluble double hydrophilic block copolymer. The polymer sensor with Schiff moiety can
sensitively, selectively and simultaneously fluorescence probe Zn2þ (green) and Pb2þ (blue) through the different
Dual-functional fluorescent sensor
Water-soluble polymer
Bioimaging
Lead
fluorescence color. The linear concentration ranges for Pd2þ and Zn2þ were 0.8–10.0
μM and 0–10.0 μM with the
detection limits of 0.7
μ
M and 0.2
μ
M, respectively. The probe responding to Naþ, Mn2þ, Fe2þ, Fe3þ, Cu2þ, Zn2þ
,
Zinc
Cd2þ, Al3þ, Cr3þ, Co2þ, Ni2þ, Agþ, Ca2þ, Kþ, Mg2þ, Ba2þ, Hg2þ, and Pb2þ displays almost no significant inter-
ference with the assay process. The sensing mechanism of the PSAM probe to Pb2þ and Zn2þ was investigated by
Job’s plot experiment, 1H NMR analysis and density functional theory (DFT), the results show that the nitrogen
–
atom of CH N and oxygen atom of OH may recognize with ions and the Job’s plots for the PSAM complexes
–
demonstrate 1:1 complexation, respectively. To achieve its application, the chemosensor is successfully applied
to monitor Pd2þand Zn2þ in tap and lake water samples, its ability to monitor free Pd2þ and Zn2þ ions in living
cells or in vivo is also validated by the intracellular imaging.
1. Introduction
headache, dizzies, constipation. Furthermore, residual lead in animal
body will cause much health problem such as arteriosclerosis, anemia,
Fluorescent sensors for selective identifying metal ions have poten-
tial application for the biological imaging and environment because of
the facile analysis, high sensitivity, real-time characteristics and espe-
cially handy operation [1–8]. Among the heavy metal cations, lead is
considered to be one of the most toxic metal ions. Importantly, it is
declared one of the 2B carcinogen by W.H.O. Besides, pigments are
connected to lead, some nations demand that the contents of lead in
pigments need be controlled within 660 ppm in their environment
standards. The concentration level of lead that will result in health issue
even risk life is different between children and adults. As for adults,
muscle paralysis, headache and memory loss, etc [9–19].
Zinc, after iron, copper and aluminum, the fourth most familiar
metal to the public, also enjoys very high status in industry, moreover,
zinc plays indispensable roles in many pathological and physiological
including regulation of metalloenzymes, gene transcription, cell
apoptosis, signal transmission [20–24]. Nevertheless, excessive zinc ions
in human body are associated with neurodegenerative disorders
[25–27]. In this context, it is essential to develop multi-functional sen-
sors for sensitive and selective detection of Pb2þ and Zn2þ
.
Accordingly, many types of fluorescent probes for detection of Pb2þ
and Zn2þ ions based on rahodamine [28,29], quinolone [30,31], 1,
8-naphthalimide [32,33], BODIPY [34,35] have been reported. As far
as we are concerned, only a few fluorescent probes are developed for
simultaneous detection of Pb2þ and Zn2þ. Joseph et al. [36] reported a
concentration level higher than 100
μg/100 mL in blood is dangerous.
Children are more allergic to lead ion, the content will decrease into 72
μ
g/mL. Once lead transports into human body, it will remain in the
system for period of time, which can lead to anemia, accompanied with
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: linnaibo@xmu.edu.cn (N. Lin), syg@dhu.edu.cn (S. Guang), hongyaoxu@163.com (H. Xu).
https://doi.org/10.1016/j.orgel.2020.105711
Received 18 September 2019; Received in revised form 27 February 2020; Accepted 8 March 2020
Available online 14 March 2020
1566-1199/© 2020 Elsevier B.V. All rights reserved.

G. Wei et al.
Organic Electronics 82 (2020) 105711
triazole-appended alkoxy as the fluorescence sensor for detection of
Pb2þ and Zn2þ in CH3CN solution based on the different excitation
wavelength. Obviously, those small molecule-based fluorescence probes
for detections of lead and zinc ions have suffered from many limitations,
such as water-insoluble, low sensitivity and poor selectivity. Moreover,
considering the bio-imaging applications, small molecule fluorometric
sensors expose a few disadvantages such as exuding from the blood
vessels and rapid elimination, which will limit the measurement times.
Polymer is a kind of novel sensor, which seems to solve the above
problems. Multifunctional fluorescence chemosensors based on amphi-
philic copolymer display high selectively and sensitivity towards Al3þ in
pure aqueous solution [6,37]. A water-soluble conjugated polymer
containing rhodamine 6G motieries exhibit high selectivity and good
reversibility toward Fe3þ in pure aqueous solutions [38]. Herein,
multi-functional polymers are promising sensors to monitor of Pb2þ and
Zn2þ in pure aqueous solution in real-time.
2.3. Sample synthesis
2.3.1. Synthesis of 4-aminobenzohydrazide (SH) (Scheme 1(a))
2.0 g (13.2 mmol) of 4-aminobenzoate was dissolved in 20 mL of
absolute ethanol, 1.0 mL (19.8 mmol) of hydrazine hydrate was drop-
wise added under N2 atmosphere, then the mixture was heated to 78 �C
under reflux for 5 h. After the solution was cooled to room temperature,
the crude product was obtained by filtering and washing with deionized
water, which was then recrystallized in ethanol. Yield (after recrystal-
lization): 1.6 g (80%). FTIR (wavenumber, cmÀ 1): νN-H, 3430, 3342,
3323, and 3230;
ν
C
O, 1670. 1H NMR (600 MHz, 298 K, DMSO‑d6): δ
–
–
(ppm), 9.43 (s, 1H), 7.63 (d, 2H), 5.62 (s, 2H), 4.43 (s, 2H). 13C NMR
(150 MHz, 298 K, DMSO‑d6): δ (ppm), 167.68, 152.53, 129.74, 121.10,
and 113.94.
2.3.2. Synthesis of (E)-4-amino-N0-(2-hydroxybenzylidene)
We fabricated a multi-responsive double fluorescence sensor (PSAM)
using hydrophilic block copolymer towards Pb2þ and Zn2þ, which
contains pendant group of schiff-based moieties (SBAC) as the ion-
sensitive receptor. For solving molecular solubility, a hydrophilic
group of the acrylamide is introduced into the polymer molecule to
achieve water solubility. The PSAM sensor shows good selective
response to Pb2þ and Zn2þ ions in pure aqueous solution with turn-on
color. Meanwhile, to further investigate sensor for detection of Pb2þ
and Zn2þ ions in practical application, we carried out the detection of
Pb2þ and Zn2þ ions in tap and lake water, as well as bio-imaging in living
cells.
benzohydrazide (SB) (Scheme 1(b))
First, 1.6 g (10.5 mmol) of 4-aminobenzohydrazide (SH), 1.3 mL
(11.0 mmol) of salicylaldehyde and 30 mL of ethanol were added to a
100 mL three-neck bottle. The mixture solution was stirred at 80 �C for 3
h. After cooling to room temperature, a pale-yellow precipitate was
obtained, which was then washed with a small amount of cool ethanol.
Yield: 86.0%. FTIR (wavenumber, cmÀ 1): νOH
,
NH, 3455, 3356, and
–
–
3281;
ν
O, 1664. 1H NMR (600 MHz, 298 K, DMSO‑d6), δ (ppm), 12.65
(s, 1H),C9.01 (s, 1H), 7.72 (d, 2H), 7.54 (dd, 1H), 5.76 (s, 1H). 13C NMR
(150 MHz, 298 K, DMSO‑d6), δ (ppm), 163.50, 158.32, 153.50, 147.81,
131.80, 130.62, 130.32, 120.11, 119.67, 119.64, 117.30, and 113.57.
2. Material and methods
2.3.3. Synthesis of (E)-4-amino-N0-(2-hydroxybenzylidene)
benzohydrazide (SBAC) (Scheme 1(c))
2.1. Materials
0.5 g (1.55 mmol) of compound SB, 0.32 mL (2.32 mmol) of trime-
thylamine, and 15 mL of NMP were added to a 100 mL three-neck bottle.
The mixture was stirred for 10 min at 0~5 �C. 0.197 mL (2.02 mmol) of
acryloyl chloride was added to the three-neck bottle with stirring at
room temperature for 15 h under N2 atmosphere. Then the solution was
added to deionized water. After the filtration and recrystallization in
All materials were commercially purchased, and utilized as received
unless otherwise stated. The chemicals used in the synthesis were
acrylamide (Alfa Aesar, 99%), 2, 20-azobis (2-methylpropionitrile)
(AIBN, Merck, 98%), hydrazine monohydrate (Alfa Aesar, 98%), sali-
cylaldehyde (Sinopharm, 98%), triethylamine (Fluka, 99%), acryloyl
chloride (Alfa Aesar, 99%), N-methyl-2-pyrrolidone (NMP, Sinopharm,
98%), ethanol absolute (VWR, 99.8%), dichloromethane (Sinopharm,
98%), tetrahydrofuran (THF, Alfa Aesar, 98%), and acetone (Sino-
pharm, 98%). All the metal salts were obtained from Sinopharm
Chemical Reagent Co., Ltd. THF and dichloromethane were distilled
from fresh sodium and benzophenone before usage.
ethanol, product (SBAC) was obtained. Yield: 0.38 g (70.5%). FTIR
(wavenumber, cmÀ 1), νOH, NH, 3450 (bb), 3320 and 3251;
ν
O, 1660.
–
–
C
1H NMR (600 MHz, 298 K, DMSO‑d6), δ (ppm), 12.01 (s, 1H), 11.32 (s,
1H), 10.11 (s, 1H), 9.01 (s, 1H), 7.97 (d, 2H), 7.90 (d, 2H), 7.57 (d, 1H),
7.34 (t, 1H), 7.02–6.93 (m, 2H), 5.93 (s, 1H). 13C NMR (150 MHz, 298 K,
DMSO‑d6): δ (ppm), 168.10, 163.20, 158.32, 148.90, 143.35, 141.11,
132.30, 130.62, 129.30, 128.20, 120.97, 120.31, 120.25 and 117.30.
ESI-MS calcd for C17H15N3O3 309.3270, found 309.3265.
2.2. Measurements and instrumentation
2.3.4. Synthesis of PSAM (Scheme 1(d))
Nuclear magnetic resonance (NMR) spectra were recorded on Bruker
AC-600 spectrometers, DMSO‑d6 and D2O were used as the solvent.
Molecular weights was measured through gel permeation chromatog-
raphy (GPC) (Shimdzu, Japan). Absorption spectra were collected on a
PerkinElmer Lambda 35 UV/vis spectrometer. Fluorescence spectra
were taken on a PE 55 fluorescence spectrometer, the slit width was 4
nm and 4 nm for excitation and emission with 316 nm of the excitation,
the emission was acquired from 350 nm to 400 nm, Stock solutions (2.0
86 mg (0.28 mmol) of compound SBAC, 0.2 g (2.8 mmol) of acryl-
amide, 1.6 mg (9.7 μmol) of AIBN, and THF (10 mL) was added into a
reaction tube equipped with a magnetic stirring bar. After stirring for 2
h at 66 �C under N2, the reaction mixture was cooled to the room tem-
perature and poured into an excess of cool acetone for the precipitation.
The precipitate was redissolved in THF and then added to cool acetone
to precipitation again for three times. PSAM was acquired as a pale-
yellow powder, yield: 70% (0.2 g). GPC results that Mn of 2.7 KDa
and MW/Mn of 1.13 (Fig. S6). The degree of polymerization (DP) of
PSAM was analyzed by 1H NMR (Table S1). Hence, the polymer was
named poly(SBAC)1-co-(AM)20. SBAC content in poly(SBAC)1-co-(AM)20
block was measured to be ~4.8% mol by using SBAC as the standard.
With the similar procedures, another poly(SBAC)1-co-(AM)50 were pre-
pared. GPC analysis revealed Mn of 4.6 KDa and MW/Mn of 1.17
(Fig. S6), respectively. SBAC content in poly(SBAC)1-co-(AM)50 block
was measured to be ~1.9 mol% for poly(SBAC)1-co-(AM)50 by using
SBAC as the standard.
mM) of metal cation salts (Zn2þ, Pb2þ, Naþ, Ba2þ, Kþ, Fe2þ, Mn2þ, Fe3þ
,
Cu2þ, Cd2þ, Al3þ, Cr3þ, Ni2þ, Agþ, Ca2þ, Co2þ, Mg2þ, Hg2þ) and our
polymer sensor were prepared in distilled water. During the test, ensure
that the pH of the solution is 7 with Tris-HCl buffer (10.0 mM, pH 7.0).
In the selectivity experiments, appropriate amounts of the metal ions
stock solutions were placed into 10.0 equiv. of the sensor stock solution.
Fluorescence and UV/vis spectroscopy were taken at room temperature.
Fourier-transform infrared (FTIR) spectra were performed using KBr
discs on a Nicolet 8700 spectrometer in the 4000–400 cmÀ 1 region.
2

G. Wei et al.
Organic Electronics 82 (2020) 105711
Fig. 1. Fluorescence spectra of (SBAC)1-co-(AM)50 with excitation wavelength of Zn2þ (316 nm) and Pb2þ (327 nm), relative fluorescence intensity (F/F0) of 0.1 g/L
(SBAC)1-co-(AM)50 (b) solution after the addition of 1.0 equiv of Zn2þ, Pb2þ, Naþ, Kþ, Ba2þ, Mn2þ, Fe2þ, Fe 3þ, Cu2þ, Cd2þ, Al3þ, Cr3þ, Co2þ, Ni2þ, Agþ, Ca2þ, Mg2þ
,
and Hg2þ. Inset of (b) is the corresponsive images under UV light.
Fig. 2. Fluorescence spectra of 0.1 g/L PSAM with different concentrations of (a) Zn2þ, and (b) Pb2þ in Tris-HCl buffer solution (pH 7.0). Inset: fluorescence intensity
(λem ¼ 362 nm) vs ion concentration.
Fig. 3. Competitive effects of (a) Zn2þ and (b) Pb2þ ion with other metal ions in 0.1 g/L of PSAM Tris-HCl buffer solution.
3

G. Wei et al.
Organic Electronics 82 (2020) 105711
2.4. Cytotoxicity assays
MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide)
assay was carried out to evaluate the cytotoxic effect of the sensor in
cells. HeLa cells were seeded in 36-well plates and incubated for 24 h to
reach ca. 75% confluence before treatment. PSAM (0, 5, 10, 15, 20, and
25 mM, 100 μL) was add to the 36-well plates, the cells were cultured for
24 h. Then, adding MTT (50 μL/well, 5 mg/ml) into each well and the
cells were cultured for 4 h. After that, adding 200
well and measuring the absorbance at 316 nm.
μl of DMSO to each
2.5. Cell imaging
�
HeLa cells were cultured by DMEM at 37 C under 5% CO2, then
stained with PSAM (50 μM) with 25 min and then washed through PBS
butter solution. The cells imaging was performed on a fluorescent
Inverted microscope (BSF-60).
3. Result and discussion
Fig. 4. The time response of 0.1 g/L of PSAM sensing 10 equiv. of Zn2þ and
Pb2þ in 10.0 mM of Tris-HCl solution (λem ¼ 362 nm for Zn2þ, λem ¼ 375 nm
for Pb2þ).
3.1. Synthesis of poly(SBAC)1-co-(AM)20 and poly(SBAC)1-co-(AM)50
We prepared PSAM containing different contents of SBAC moiety
through radical polymerization. The structural characterization of the
copolymer and the data of GPC and 1H NMR were shown in Figs. S1–5
and Table S2. The percentage content of chromophore in poly(SBAC)1-
co-(AM)20 and poly(SBAC)1-co-(AM)50 was determined to be ~4.8 mol%
and ~1.9 mmol% by 1H NMR (Table S1), respectively.
3.2. Fluorescence spectroscopy of PSAM for Pb2þ and Zn2þ
The fluorescent sensing ability of PSAM was investigated in presence
of various metals of 100 μM in Tris-HCl buffer solution (pH ¼ 7.0) at the
room temperature. PSAM of 0.1 g/L without metal ions has a low
fluorescence emission peak with excitations of 316 nm (Zn2þ) and 327
nm (Pb2þ). After the addition of 1.0 equiv. different metal cation ions
such as Naþ, Ba2þ, Mn2þ, Fe3þ, Cu2þ, Zn2þ, Fe2þ, Cd2þ, Al3þ, Cr3þ
,
Co2þ, Ni2þ, Agþ, Kþ, Ca2þ, Mg2þ, Hg2þ, Pb2þ to the solutions of PSAM,
no or a very weak fluorescence was observed except for Pb2þ and Zn2þ
.
Because the less solubility of (SBAC)1-co-(AM)20 in pure water leads to
the low fluorescence intensity of the complex (Fig. S9), other metal ions
have relatively large interference in detecting Zn2þ and Pb2þ. (SBAC)1-
co-(AM)50 is selected for detailed study. The Zn2þ and Pb2þ ions cause
remarkable fluorescence enhancements at 362 nm and 375 nm
compared with (SBAC)1-co-(AM)50. (Fig. 1a and b). Two different
Fig. 5. Fluorescence intensities of 0.1 g/L PSAM with 10.0 equiv. Zn2þ/Pb2þ at
different pH (1–13), respectively (λex ¼ 362 nm for Zn2þ, λex ¼ 375 nm
for Pb2þ).
Fig. 6. Job plot for plot PSAM with (a) Zn 2þ (b) Pb 2þ in water solutions. The total concentration on plot PSAM and Zn2þ/Pd2þ is 10
μM, respectively. (0.1 g/L water
solution of PSAMs, [SBAC] ¼ 7.6
μ
M; 10.0 mM Tris-HCl, pH 7.0; λex ¼ 362 nm for Zn2þ, λex ¼ 375 nm for Pb2þ, slit widths: λem. 4.0 nm, λem. 4.0 nm).
4

G. Wei et al.
Organic Electronics 82 (2020) 105711
Fig. 7. (a) Partial 1H NMR spectra of PSAM in D2O alone and in the presence of Pb2þ and Zn2þ, (b) HOMO and LUMO orbitals of SBAC-Pb2þ, SBAC-Zn2þand SBAC.
emissions wavelengths indicate that PSAM could be utilized as a dual-
channel fluorescent sensor for Zn2þ and Pb2þ in the completely water
solution medium. The difference in fluorescence emission peak for
sensing the Zn2þ and Pb2þ is due to the different energy gaps of two
complex, PSAM-Zn2þ and PSAM-Pb2þ [39].
other relevant metal cation ions, such as Zn2þ, Pb2þ, Naþ, Ba2þ, Fe2þ
Kþ, Fe3þ, Cu2þ, Cd2þ, Al3þ, Cr3þ, Co2þ, Mn2þ, Ni2þ, Agþ, Ca2þ, Mg2þ
,
,
Hg2þ. As depicted in Fig. 3, Zn2þ/Pb2þ detection was not disturbed via
the presence of detected metal cation ions except Fe3þ, Fe2þ and Cu2þ
,
due to the strong binding of Cu2þ, Fe2þ and Fe3þ ions with PSAM. PSAM
could be as a fluorescence probe for Zn2þ and Pb2þ in the presence of
metal ions. The fluorescent emission peak intensity at 362 nm for Zn2þ
hited the F/F0 ¼ 18 and at 375 nm of Pb2þ was F/F0 ¼ 14. The emission
intensity enhancement at 362 nm for Zn2þ hits the highest F/F0 ¼ 18 and
the emission intensity enhancement at 375 nm of Pb2þ was F/F0 ¼ 14.
Therefore, we could differentiate the blue and green fluorescent of Zn2þ
and Pb2þ by the different fluorescence color.
The emission spectra of PSAM with different concentrations of Zn2þ
and Pb2þ in Tris-HCl buffer solution (pH ¼ 7.0) were illustrated in Fig. 2.
As the concentration of Pb2þ and Zn2þ increased, the fluorescence
emission intensity increased until 2.0 equiv. and then there was no
further change. The linear concentration ranges for Pd2þ and Zn2þ were
0.8–10.0 μM and 0–10.0 μM with the detection limits of 0.7 μM and 0.2
μ
M, respectively (Fig. S6). Based on Benesi-Hildebrand, the complex
constants of PSAM with Pb2þ and Zn2þwere 4 � 104 MÀ 1 and 4 � 105
Response time for the designed sensor is as important as sensitivity
and selectivity. The response time of PSAM (0.1 g/L) for sensing 1.0
equiv of Zn2þ/Pb2þ in buffer solutions was studied (Fig. 4). The fluo-
MÀ 1 respectively. (Fig. S7).
To further evaluate the practical applicability of PSAMs as a selective
fluorescence sensor for Zn2þ and Pb2þ, competitive experiments were
carried out in the presence of Zn2þ/Pb2þ of 0.1 g/L with 1.0 equiv. of
rescence analysis displays that the detection between PSAM and Zn2þ
/
Pb2þ starts immediately with gently shaking. The fluorescence emission
Scheme 1. Synthesis schematic of the chemical sensor PSAM.
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G. Wei et al.
Organic Electronics 82 (2020) 105711
Scheme 2. Proposed binding mode of PSAM with Pb2þ and Zn2þ
.
peak intensity of PSAM in the presence of Zn2þ/Pb2þ increases in Tris-
HCl buffer solution in ~15 min, reaches strongest value in 40 min,
after that tends to be stable. We can conclude that PSAM and Zn2þ/Pb2þ
complete coordination in 39 min and 30 min, respectively.
The effect of pH on the detection of PSAM was also tested under
various pH conditions. From Fig. 5, the fluorescence emission intensity
of PSAM no changes in the pH range from 2.0 to 13.0. The pH of the
PSAM solution altered by adding of Trils-HCl buffer solution. In the pH
range of 7.0–9.0, PSAM displays a high fluorescence response toward
Zn2þ/Pb2þ, demonstrating that the sensor could be used under the
biological and environmental condition. For examples, the fluorescence
sensor of PSAM could be utilized to be monitor Zn2þ/Pb2þ in water and
living cells, the pH range of which is usually found to be 6.0–7.6 [40].
Fig. 8. Bright and fluorescence images of HeLa cells after incubating with
PSAM (50
μ
mol/L), PSAM þ Zn2þ, and PSAM þ Pb2þ for 0.5 h.
using Job’s plot. From Fig. 6, the Job’s plots for the PSAM complexes
demonstrate 1:1 complexation stoichiometry, respectively.
To evaluate the utility of copolymer fluorescent sensor, PSAM was
used as a fluorescent probe to measure Zn2þ and Pb2þ in bottled water
and tap water. No detectable of Zn2þ or Pb2þ residues was found in the
actual samples prior to the addition of Zn2þ and Pb2þ. Recovery tests
were tested from spiked with 85–93% Zn2þ or Pb2þ. The results were
showed in Table 1. As we known, the recovery rate should be limited
between 95%~105%, and relative standard deviation (RSD) should be
controlled below 5%. The recovery rate of Zn2þ ranges from 95.73% to
101.43% and recovery rate of Pb2þ ranges from 96.32% to 102.58%.
Those results display that the method can provide a potentially efficient
detection system for Zn2þ or Pb2þ in actual samples.
To elucidate the probing mechanism of PSAM toward Zn2þ and Pb2þ
,
titration experiments of PSAM with Zn2þ and Pb2þ were carried out
based on 1H NMR. The 1H NMR spectrum of PSAM in D2O and detailed
assignment of each proton was depicted in Fig. 7a. The chemical shifts at
9.87, 7.72, 7.52–6.50, 3.78, 3.65 and 2.30–1.25 ppm are assigned to the
proton Hm, Hi, He, Hnþlþkþjþgþf, Hc, Hd, Haþb of PSAM, respectively.
Upon addition of Zn2þ, not only chemical shift of Hm at 9.87 ppm dis-
appears, but also that of Hi shifts from 7.72 to 8.14 ppm, indicating that
–
the nitrogen atom of CH N and oxygen atom of OH may recognize with
–
Pb2þ. While upon addition of Pb2þ, Hm peak at 9.87 ppm also disap-
pears, Hi peak alters from 7.72 to 8.31 ppm. For further verifying the
probing mechanism of PSAM toward Zn2þ and Pb2þ, energy-optimized
structures of monomer SBAC and its complexes are calculated by den-
sity functional theory (DFT) at the B3LYP22/LanL2DZ level using the
program Gaussian 09 (Fig. 7b). The energy gaps (ΔE) between HOMO
and LUMO for SBAC, SBAC-Zn2þ and SBAC-Pb2þ are 4.12, 3.28 and 3.72
eV respectively. A low ΔE well explains why there are strong spectral
absorption and fluorescent emission at long-wavelength for SBAC in the
presence of Zn2þ and Pb2þ. Compared with that of SBAC-Pb2þ, the lower
ΔE of SBAC-Zn2þ hints the longer fluorescence emission wavelength.
According to above results, the probable binding modes of PSAM with
Pb2þ and Zn2þare proposed in Scheme 2.
The modes between SBAC and the ions, Pb2þ and Zn2þ were tested by
Table 1
Fluorescent determination results of Zn2þ and Pb2þ in real samples.
Extract
Plus
Measure
Recovery
rate/%
Relative standard
deviation (RSD)/%
sample
scalar/
μ
M
value/μM
For Zn2þ
Tap
0.40
0.50
0.60
0.40
0.50
0.60
3.00
4.00
5.0
0.39
0.51
0.57
0.43
0.49
0.61
3.13
3.75
5.27
2.87
4.19
5.24
97.50
0.03
0.05
0.02
0.04
0.06
0.03
0.02
0.04
0.05
0.06
0.03
0.04
100.20
95.00
water
Lake
107.50
98.00
water
101.17
104.33
93.75
For Pb2þ
Tap
water
Lake
105.40
95.67
3.00
4.00
5.00
water
104.48
104.80
Fig. 9. Fluorescence imaging of Zn2þ and Pb2þ in zebrafish with PSAM (scale
bar ¼ 1000 μM).
6

G. Wei et al.
Organic Electronics 82 (2020) 105711
Fig. 10. a. Representative photographs of mice injected with PSAM and with or without Zn2þ b. H&E-stained major organs (heart, liver, spleen, stomach, kidney and
lung) in mice injected with PBS (control) and PSAM after 7 days (scale bar ¼ 50 μM).
The cytotoxicity of PSAM with the different concentrations of from
staining. Compared with the control group, no pathological changes to
0–20
μ
M were conducted with living HeLa cells via MTT assay (Fig. S8).
the organs are observed (Fig. 10b).
The result showed that the copolymer is not toxic to cells. Cells were
�
cultured with 50
μ
M of PSAM for 25 min at 37 C under 5% CO2 for
4. Conclusions
cellular fluorescence imaging. As depicted in Fig. 8, no fluorescence
colour was found in the cell image without Zn2þ/Pb2þ (Fig. 8b). Here-
In summary, the copolymer of PSAM is synthesized, which can serve
as a dual-channel sensor to Pb2þ and Zn2þ, respectively. The PSAM
based on Schiff segment can selectively detect Pb2þ (blue) and Zn2þ
(green) without interference by other metal ions. Real sample analysis
demonstrates that PSAM can be applied to a recognition system for Pb2þ
and Zn2þ in natural water samples, showing with good selectivity and
sensitivity. Finally, we realize the application of PSAM for monitoring
Pb2þ and Zn2þ ions in vivo and in vitro. This work will provide inspi-
ration for the development of new multi-channel water-soluble chemical
sensors.
after, the cells continued to culture 25 min with 50
μ
M of Zn2þ/Pb2þ at
37 �C and then were washed three times with PBS. Two fluorescent
colors (blue and green) were found from the cell imaging, the cells with
Zn2þ emit green fluorescence (Fig. 8e and f), while the cells with Pb2þ
emit blue fluorescence (Fig. 8h and i). We can conclude that the probe of
PSAM is a suitable and biocompatible probe for detecting Zn2þ and Pb2þ
ions in living cells.
To demonstrate that the imaging of living animals could be realized
for sensing Zn2þ and Pb2þ by PSAM, the zebrafish was employed to
investigate the capacity of PSAM to monitor Zn2þ and Pb2þ. Zebrafish
purchased from Shanghai GeneBio Co., Ltd. Five groups of zebrafishes
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgements
were co-incubated with 0
μ
M (without Zn2þ and Pb2þ), 5
μM and 20
μ
μ
M
M
of Zn2þ, 5
μM and 20
μ
M of Pb2þ for 20 min, and then treated with 20
PSAM for further 20 min. As showed in Fig. 9, green and blue fluores-
cence signal of zebrafish cannot be collected under green or blue
channel without metal ions. With the gradual addition of metal ions, a
striking enhancement of fluorescence signal in the green or blue channel
was collected, confirming that Zn2þ and Pb2þ can be specifically
recognized by PSAM in zebrafish. That implies that PSAM is capable of
entering zebrafish and sensing Zn2þ and Pb2þ efficiently.
This project supported by the National Natural Science Foundation
of China (Grant Nos. 21671037, 21771036), Fujian Provincial Depart-
ment of Science and Technology (Grant No. 2017J06019).
Motivated by its remarkable in vitro imaging performance, we
further evaluate the feasibility of using PSAM as in vivo imaging. Due to
the toxicity of lead ions, we only made zinc ions in animal experiments.
All animal experiments are performed in compliance with the Animal
Management Rules of the Ministry of Health of the People’s Republic of
China (Document no. 55. 2001) and the guidelines for the Care and Use
of Laboratory Animals of China Pharmaceutical University. As shown in
Fig. 10a, when PSAM is injected without Zn2þ, there is no fluorescence.
As the concentration of zinc ions increasing, the fluorescence range in-
creases, the fluorescence image range gradually spreads. It shows that
PSAM has good imaging effect and no toxicity in vivo. Non-toxicity of
PSAM is further supported by the H&E. To evaluate the systemic toxicity
of PSAM, the mice are sacrificed at the 7th day and their major organs
(heart, liver, spleen, stomach, kidney and lung) are excised for H&E
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.orgel.2020.105711.
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