A water-soluble molecular probe with aggregation-induced emission for discriminative detection of Al3+ and Pb2+ and imaging in seedling root of Arabidopsis

Article abstract of DOI:10.1016/j.saa.2019.117335

Luminogens with aggregation-induced emission (AIE) have been used to develop a new type of molecular probes based on analyte-triggered aggregation, but it still remains a challenge to design water-soluble AIE-active probe for specific detection of metal i

Full text of DOI:10.1016/j.saa.2019.117335

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A water-soluble molecular probe with aggregation-induced emission for
discriminative detection of Al3+ and Pb2+ and imaging in seedling root
of Arabidopsis
1
1
⁎
Pengfei Xu , Zhiyi Bao , Chenyi Yu, Qianqian Qiu, Mengru Wei, Wenbin Xi, Zhaosheng Qian, Hui Feng
Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2019
Received in revised form 18 June 2019
Accepted 30 June 2019
Available online 02 July 2019
Luminogens with aggregation-induced emission (AIE) have been used to develop a new type of molecular probes
based on analyte-triggered aggregation, but it still remains a challenge to design water-soluble AIE-active probe
for specific detection of metal ions. Herein, we designed and synthesized a water-soluble molecular probe with
AIE property for discriminative detection of aluminum ion and lead ion. Four carboxylic acid groups were incor-
porated into a tetraphenylethylene unit to enhance the coordination affinity and increase water-solubility in
aqueous solution. The designed probe can be selectively lighted up by aluminum ion and lead ion via
coordination-triggered AIE process. Discrimination of aluminum ion and lead ions based on the probe can be
achieved in quantitative manner with the assistance of suitable masking reagents. This probe was further used
to image aluminum ions in living cells of seedling roots of Arabidopsis, and the results showed that this probe
is capable of imaging aluminum ions in living cells avoiding the interference of lead ions, and is suited for
long-term imaging due to its excellent photostability. This work expands the application scope of AIE-active
probes in discriminative detection of metal ions, and provides a design direction for water-soluble AIE probes
to avoid the false signals from self-precipitation under physiological conditions.
Keywords:
Aggregation-induced emission (AIE)
Molecular probe
Aluminum ion
Lead ion
Imaging
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
caused by aggregation and significant photobleaching under the long-
term irradiation of excitation light.
The accurate measurement of metal ions is a persistent challenge for
chemists because some lighter metal ions play indispensible roles in
diverse biological processes while exposure to heavier and toxic metal
ions has increased with industrialization. Aluminum as the third most
abundant element in the earth crust, has continuously entered eco-
sphere and biosphere due to the increase of acid rains [1]. Present evi-
dence has indicated that the intake of aluminum ion causes a
significant toxicity to organisms including plants and human [2,3].
Moreover, lead ion is another significant pollutant occurred widely in
environments because of its highly toxic feature to organisms [4,5].
The quantification and imaging of these toxic metal ions generally
require accurate and specific optical methods relying on fluorogenic
indicators [6,7]. Despite diverse fluorescent probes have been devel-
oped for Al3+ and Pb2+, most of them were based on conventional
dyes with aggregation-induced quenching (ACQ) feature. These ACQ
fluorophores frequently suffer from an appreciable decline in brightness
Aggregation-induced emission luminogens (AIEgens) have shown
great power and promising prospect in sensing and imaging since its
discovery by Tang group [8,9]. In contrast with ACQ fluorogens, AIEgens
have weak emission in dissolved state, but emit intense fluorescence in
aggregated or solid state [10]. This unique property of AIEgens allows to
establish a novel type of detection strategy based on aggregation-
induced emission [11,12], and these AIE-active probes exhibit better
photostability in long-term imaging than existing ACQ probes [13]. As
a result, a variety of AIE-active probes for detecting metal ions including
Ag+ [14,15], Hg2+ [16,17], Zn2+ [18–21], and Ca2+ [22,23] based on
coordination-triggered aggregation processes have been developed,
and a number of fluorometric methods for Al3+ based on diverse
AIEgens have also reported [24–31]. However, most of these reported
AIE-active molecular probes have poor water-solubilities, and thus spe-
cific organic solvents such as DMSO and THF are required for them to
quantify and image Al3+ because of their poor solubility in water. The
low solubility of AIE probes in aqueous solution probably causes false
signals from their self-precipitation inside live cells during the imaging
process, which will greatly impact the sensitivity and accuracy of track-
ing Al3+ in biosystems. In addition, AIE-active probes for Pb2+ are very
⁎
Corresponding author.
E-mail address: fenghui@zjnu.cn (H. Feng).
These authors contributed to this work equally.
1
https://doi.org/10.1016/j.saa.2019.117335
1386-1425/© 2019 Elsevier B.V. All rights reserved.

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P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
rare currently [32–34], and dual-functional indicators for discriminative
quantitation of Al3+ and Pb2+ in a facile way have not reported till now.
In this contribution, we designed and synthesized a water-soluble
molecular probe for quantification of Al3+ and Pb2+ based on ion-
triggered aggregation-induced emission. Four carboxylic acid groups
were incorporated into a tetraphenylethylene unit in the probe. The in-
troduction of these carboxylic acid groups not only endows excellent
solubility of the probe in water but also provides the recognition units
for target metal ions. As shown in Scheme 1, the probe TPE-4CO2Na
can be effectively assembled by Al3+ and Pb2+ via their coordination re-
actions between metal ions and carboxylic acid groups. With the assis-
tance of masking agents, the probe TPE-4CO2Na was used for
discriminative detection of Al3+ or Pb2+ in a fluorescence turn-on man-
ner. The imaging performance of the probe for Al3+ in live cells was fur-
ther evaluated in Arabidopsis thaliana.
as the eluent. Compound 2 was obtained in 70% yield (1.01 g). 1H
NMR (600 MHz, CDCl3) δ (ppm) 6.93 (d, J = 6 Hz, 8H), 6.64 (d, J =
6 Hz, 8H), 3.77 (s, 12H). 13C NMR (150 MHz, CDCl3) δ(ppm) 158.96,
138.08, 138.07, 133.75, 114.19, 56.28. HRMS (ESI) m/z: [M + K+
]
491.1633, (calcd. for C30H28O4, 452.0653).
2.3. Synthesis of 1,1,2,2-tetrakis(4-hydroxyphenyl)ethylene (Compound 3)
A certain amount of BBr3 (4 mL, 44.2 mmol) was first added in 50 mL
of dry DCM solution containing Compound 2 (2.00 g, 4.42 mmol) at
−20 °C. The mixture was stirred for 40 h at room temperature, and
then was concentrated under vacuum. The mixture was poured into
100 mL water and stirred for 5 min after adding 3 ml ethanol, and
then massive white precipitates were generated. The final product
was obtained in 97% yield (1.7 g) after filtration and drying. 1H NMR
(600 MHz, DMSO-d6) δ 9.24 (s, 4H), 6.70 (d, J = 12 Hz, 8H), 6.48 (d, J
= 12 Hz, 8H). 13C NMR (150 MHz, DMSO-d6) δ 155.34, 137.67,
135.05, 131.94, 114.47. HRMS (ESI) m/z: [M + Na+] 419.1249, (calcd.
for C26H28O4, 396.1349).
2. Experimental
2.1. Materials and reagents
Triple-distilled water was utilized throughout the whole experimen-
tal process. 4,4-Dimethoxybenzophenone, Zn dust, boron tribromide
and ethyl chloroacetate were purchased from Sigma-Aldrich Company
(Shanghai, China). HEPES solution (10 mM, pH 7.0) was used as the
buffer solution. All reagents were of analytical grade and without any
further purification.
2.4. Synthesis of tetraethyl 2,2′,2″,2‴-((ethane-1,1,2,2-tetrakis(benzene-
4,1-diyl))tetrakis-(oxy)tetraacetate (Compound 4)
Compound 3 (1.5 g, 3.78 mmol), Cs2CO3 (8.631 g, 26.49 mmol) and
ethyl chloroacetate (3.142 mL, 26.49 mmol) was added in 80 mL DMF,
and then the mixture was stirred for 48 h at 80 °C. White precipitates
were generated after the mixture was poured into water. The final prod-
uct was obtained in 90% yield (2.57 g) after washing and drying. 1H
NMR (600 MHz, DMSO-d6) δ 6.85 (d, J = 12 Hz, 8H), 6.69 (d, J =
12 Hz, 8H), 4.68 (s, 8H), 4.15 (q, J = 18 Hz, 8H), 1.18 (t, J = 12 Hz,
12H). 13C NMR (150 MHz, DMSO-d6) δ 168.68, 155.93, 138.20, 136.77,
2.2. Synthesis of 1,1,2,2-tetrakis(4-methoxyphenyl)ethene (Compound 2)
A typical procedure for synthesis of Compound 2 was as follows. Zn
dust (1.84 g, 28 mmol) and TiCl4 (1.58 mL, 14 mmol) were refluxed for
2 h in 50 mL of dry THF under N2 atmosphere. A solution of 4,4-
dimethoxybenzophenone (1.50 g, 7.0 mmol) in dry THF (20 mL) was
added to the preceding suspension, and then the reaction was refluxed
at 80 °C for 12 h. An aqueous solution containing 10% K2CO3 (50 mL)
was added after the reaction mixture was cooled down to room temper-
ature. The resulting product was extracted with ethyl acetate. The sol-
vent was evaporated under vacuum and the crude product was
purified by a silica gel column using hexane-ethyl: acetate (1:1, v/v)
131.95, 113.86, 64.60, 60.64, 14.02. HRMS (ESI) m/z: [M + Na+
]
763.2723, (calcd. for C42H44O12, 740.2823).
2.5. Synthesis of sodium 2,2′,2″,2‴-((ethane-1,1,2,2-tetrakis(benzene-4,1-
diyl))tetrakis-(oxy)tetraacetate (Compound 5, TPE-4CO2Na)
Compound 5 was synthesized by the reaction between Compound 4
(1.3 g, 1.75 mmol) and NaOH (0.28 g, 7 mmol) in 40 ml methanol. The
Scheme 1. Schematic Illustration of Discriminative Detection of Al3+ and Pb2+ Based on Aggregation-Induced Emission of the Probe TPE-4CO2Na with Assistance of Masking Agents.

P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
3
Scheme 2. Synthetic Route of the Probe TPE-4CO2Na.
white precipitates were collected after filtration and drying (95% yield).
1H NMR (600 MHz, D2O) δ 7.03 (d, J = 12 Hz, 8H), 6.72 (d, J = 6 Hz, 8H),
4.40 (s, 8H). 13C NMR (150 MHz, D2O) δ 176.72, 156.12, 138.81, 137.29,
132.45, 113.74, 66.65. HRMS (ESI) m/z: [M-H+] 627.1477, (calcd. for
experiment was performed on a Leica TCS SP5 model confocal laser
scanning microscope (Germany) with an excitation at 355 nm and
a variable bandpass emission filter (475–530 nm). Five-day-old
seedlings with healthy roots were divided into three groups. The
first group was used as mock control. The second group was first
treated with 0.5 mM TPE-4CO2Na solution for 10 min, and then incu-
bated in an aluminum ion solution (0.5 mM) for 10 min after these
seedlings were washed with triple-distilled water. The third groups
were treated with 0.5 mM TPE-4CO2H in DMSO/water mixed solu-
tion for 30 s, and then incubated in an aluminum ion (or lead ion)
solution (0.5 mM) for 10 min after washing. All groups were imaged
using the confocal laser scanning microscope at the emission range
of 475–530 nm. All the photostablity experiments were carried out
under the radiation of 355 nm and the irradiation time of 1, 3 and
5 min, respectively.
C34H28O4, 628.1557).
2.6. Discriminative detection of aluminum ion and lead ion based on TPE-
4CO2Na
For quantitative detection of Al3+, a TPE-4CO2Na solution with a fixed
concentration (20.0 μM) in HEPES buffer (pH 7.0) was first prepared, and
then different amounts of Al3+ in the range of 0.0–650.0 μM were sepa-
rately added into the preceding TPE-4CO2Na solution. The PL spectra of
the resulting mixtures were recorded at the excitation of 355 nm using
a xenon arc lamp. The quantitative detection of Pb2+ was performed in
a similar procedure using Pb2+ instead of Al3+, and the used amount of
Pb2+ was in the range of 0.0–500.0 μM. Selectivity test of the assay to
Al3+ and Pb2+ was conducted as follows. A certain amount of each
chosen metal cation (100.0 μM) including K+, Mg2+, Ca2+, Ba2+, Al3+
,
Pb2+, Cd2+, Co2+, Fe2+, Ag+, Zn2+, Hg2+, Cu2+, Mn2+, Cr3+ and Fe3+
was separately added into a TPE-4CO2Na solution (20.0 μM) in HEPES
buffer, and then the resulting solutions were monitored using fluores-
cence spectrometer at 480 nm emission. For the discriminative detection
of aluminum ion and lead ion, the protocol is same to the normal detec-
tion except for the addition of masking reagents. Glutathione and NaBF4
were separately used to mask the effect of lead ion and aluminum ion,
and their concentrations were 100.0 μM. All the detections were
repeated at least three times.
2.7. Live-cell imaging of aluminum ions in root cells of Arabidopsis thaliana
The wild-type (Col-0) seeds of Arabidopsis thaliana were surface
sterilized and imbibed for 3 days at 4 °C in dark and then sown onto
0.5 × Murashige & Skoog (MS) 1.5% (w/v) agar plates. Seedlings were
vertically grown on plates in a climate-controlled growth room (22/20
°C day/night temperature, 16/8-h photoperiod, and 80 μE s−1 m−2
light intensity). Five-day-old seedlings with healthy roots were used
in this study unless otherwise specified. Fluorescence imaging
Fig. 1. PL spectra of an aqueous solution and powder of TPE-4CO2Na. Inset: A photoimage
of TPE-4-CO2Na powder under UV light.

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P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
Fig. 2. (A) PL spectra of an aqueous solution of TPE-4CO2Na in the presence of different amounts of THF. Inset: Corresponding fluorescence images. (B) PL spectra of an aqueous solution of
TPE-4CO2Na at different pHs from 1.0 to 5.0. Inset: Corresponding fluorescence images.
3. Results and discussion
Dimethoxybenzophenone (1) was used as the starting material to syn-
thesize 1,1,2,2-tetrakis(4-methoxyphenyl)ethylene (2) via a McMurry
coupling reaction. 1,1,2,2-Tetrakis(4-hydroxyphenyl)ethylene (3) was
generated by the following conversion of hydroxyl groups from
methoxy groups through the treatment of BBr3. Four carboxylic esters
were further introduced via the nucleophilic substitution between
(3) and ethyl chloroacetate to produce (4). The probe TPE-4CO2Na
(5) was finally acquired by the hydrolysis of 4 using NaOH. All the inter-
mediates and the final product were obtained in good yields, and all of
3.1. Synthesis and characterization of the probe TPE-4CO2Na
Tetraphenylethylene as a typical AIE fluorophore was adopted as the
signaling unit, and carboxylic acid groups acted as the recognition unit
in the probe. Four carboxylic acid groups were introduced to the
probe to increase the solubility of the designed probe in water.
Scheme 2 shows the synthetic route of the probe TPE-4CO2Na. 4,4′-
Fig. 3. (A) PL spectra of a HEPES solution of TPE-4CO2Na (20.0 μM) in the presence of different metal ions (500.0 μM). Inset: Corresponding fluorescence images. (B) The enhancement
ratios I/I0 versus metal ions. (C) SEM image of TPE-4-CO2Na (20.0 μM) in the presence of Al3+ (500.0 μM). (D) SEM image of TPE-4CO2Na (20.0 μM) in the presence of Pb2+ (500.0 μM).

P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
5
them were fully purified and characterized using 1H NMR, 13C NMR and
MS (Figs. S1–S12).
weakly emissive at pH N 5.0, but the fluorescence of the TPE-4CO2Na so-
lution was progressively intensified as the decrease of pH from pH 5.0 to
pH 1.0. The pH-triggered fluorescence enhancement was due to the ag-
gregation of protonated form of TPE-4CO2Na. In comparison with pre-
ceding AIE probes [24–31], TPE-4CO2Na remained very weakly
emissive in neutral water without the assistance of organic solvents.
The PL spectra and lifetime of the acid form of the probe were also ex-
amined for comparison. Fig. S15 showed that the emission maximum
of TPE-4CO2H was relatively blue-shifted to 436 nm, and its lifetime
(2.2 ns) was comparable to that of the probe. These observations dem-
onstrated that TPE-4CO2Na exhibited prominent AIE behavior and had
excellent water solubility, avoiding false signals from self-precipitation
in aqueous solution.
The probe TPE-4CO2Na can dissolve very well in water, but hardly
dissolve in common organic solvents such as THF. An aqueous solution
of TPE-4CO2Na only has a very weak blue fluorescence, which is hardly
observed by naked eye. Its fluorescence spectrum in Fig. 1 shows that its
emission maximum is 400 nm when excited by the optimum excitation
wavelength of 264 nm. Its lifetime was determined to be 0.7 ns from the
time-resolved PL decay curve in Fig. S13. In contrast to its dissolved
state, the solid powder of TPE-4CO2Na emits intense green light under
the UV light. An apparent red-shift in emission maximum for TPE-
4CO2Na powder was observed relative to that in dissolved state. The
lifetime at 468 nm was lengthened to 2.1 ns determined from the
time-resolved PL decay curve in Fig. S14. The quantum yield of TPE-
4CO2Na powder was determined to 0.25 using an absolute method.
The much higher emission efficiency of the solid state than the dissolved
state provides an indication that TPE-4CO2Na possesses a significant AIE
behavior. This AIE property was further verified by aggregation pro-
cesses caused by a poor solvent and pH change in aqueous solution. A
gradual enhancement trend in PL intensity of TPE-4CO2Na solutions
was observed in Fig. 2A as the amount of THF in the mixed solutions
was increased from 0% to 98%. As the continuous addition of THF into
an aqueous TPE-4CO2Na solution, the solutions became more and
more turbid accompanying more and more intense fluorescence, clearly
suggesting unique AIE property of TPE-4CO2Na. The influence of TPE-
4CO2Na by pH was shown in Fig. 2B. The TPE-4CO2Na solution remained
3.2. Discriminative detection of Al3+ and Pb2+ based on ion-triggered AIE
The PL responses of TPE-4CO2Na to 16 commonly used metal ions
including K+, Mg2+, Ca2+, Ba2+, Al3+, Pb2+, Cd2+, Co2+, Fe2+, Ag+
,
Zn2+, Hg2+, Cu2+, Mn2+, Cr3+ and Fe3+ were first assessed as shown
in Fig. 3A. Only the addition of Al3+ (650.0 μM) or Pb2+ (500.0 μM)
caused a significant PL enhancement of TPE-4CO2Na, and a bright
green emission can be readily observed by naked eye after the introduc-
tion of Al3+ or Pb2+. The equivalent amount of Al3+ resulted in much
brighter fluorescence than Pb2+, which was clearly reflected by their re-
spective PL enhancing ratios (186 for Al3+, 111 for Pb2+) in Fig. 3B. The
quantum yield of TPE-4CO2Na after binding with Al3+ was determined
Fig. 4. (A) PL spectra of the probe TPE-4CO2Na in the presence of different components: (a) the probe (20.0 μM); (b) the probe (20.0 μM) and GSH (100.0 μM); (c) the probe (20.0 μM), GSH
(100.0 μM) and Pb2+ (100.0 μM); (d) the probe (20.0 μM), GSH (100.0 μM), Pb2+ (100.0 μM) and Al3+ (100.0 μM). (B) PL spectra of the probe TPE-4CO2Na in the presence of different
components: (a) the probe (20.0 μM); (b) the probe (20.0 μM) and NaBF4 (100.0 μM); (c) the probe (20.0 μM), NaBF4 (100.0 μM) and Al3+ (100.0 μM); (d) the probe (20.0 μM),
NaBF4 (100.0 μM), Al3+ (100.0 μM) and Pb2+ (100.0 μM). (C) PL spectra versus the concentration of Al3+ from 0.0 to 650.0 μM. (D) PL spectra versus the concentration of Pb2+ from
0.0 to 500.0 μM.

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P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
to be 25.7%, which was 180 times higher than the quantum yield of TPE-
4CO2Na in solution (0.14%). We assumed that these PL enhancements
caused by Al3+ and Pb2+ were due to the generation of aggregates be-
tween metal ions and TPE-4CO2Na via coordination. This assumption
was verified by SEM images in Fig. 3C and D, where a great deal of
large aggregates in millimeter magnitude was generated. These results
provided solid evidence that the presence of Al3+ or Pb2+ triggered the
generation of large aggregates of TPE-4CO2Na with intense green fluo-
rescence, and offered the opportunity to establish quantitative detec-
(6.2–21.6 nM) [29], but is better than those of most approaches based
on AIEgens (1.5–5.3 μM) [24,25,27,30] and ACQ fluorophores (1.0–21.7
μM) [35–38]. The appreciable decline in response sensitivity of our
method is due to the large solubility of TPE-4CO2Na in water, and this
sacrifice in sensitivity is acceptable under the premise of avoiding the
false signal from self-precipitation due to low water-solubility. A similar
PL increase trend with the concentration of Pb2+ from 0.0 to 500.0 μM
was shown in Fig. 4D, and calibration curve between PL intensity and
Pb2+ concentration ranging from 2.0 to 300.0 μM was acquired
(Fig. S17). The detection limit for Pb2+ was calculated to be 0.6 μM,
and this value is comparable to those of fluorescent methods in the liter-
ature [7,39].
tion method of Al3+ and Pb2+
.
Because both Al3+ and Pb2+ are capable of lighting up the probe TPE-
4CO2Na, mutual interference can occur during the detection. As a result,
we attempted to use masking reagents to minimize mutual interference
between them. It was found that glutathione can tightly bind to Pb2+ but
exerts no significant interaction with Al3+, and thus glutathione was
chosen as an effective masking agent to avoid the interference from
Pb2+ during the detection of Al3+. Fig. 4A shows the addition of single
glutathione or the equivalent of glutathione and Pb2+ causes no PL re-
sponse, but continuous addition of the same amount of Al3+ results in
a sharp increase of PL intensity. This indicates that the introduction of
glutathione effectively masks the added Pb2+ without any impact on
Al3+-triggered PL enhancement. In the same way, we found that NaBF4
3.3. Live cell imaging of aluminum ions in Arabidopsis thaliana
Discriminative detection of aluminum ions over lead ions in the
presence of glutathione based on TPE-4CO2Na enables us to specifically
image Al3+ in live cells because of the occurrence of a high amount of
glutathione inside living cells [40,41]. Arabidopsis thaliana as a typical
model plant was selected to examine the imaging performance of
TPE-4CO2Na in live cells [42,43]. Five-day old seedlings of Arabidopsis
thaliana was used because their transparent roots are suited for the pen-
etration of visible excitation and emission light. We first assessed the
cell permeability and imaging ability of water-soluble TPE-4CO2Na. As
shown in Fig. S18, the root cells of Arabidopsis thaliana seedling treated
with TPE-4CO2Na exhibits a very weak autofluorescence as same as the
mock control has, but an apparent pseudo green color from the root tips
can be observed after continuous treatment of the seedlings with TPE-
4CO2Na and Al3+. This faint fluorescence signal implies that water-
soluble TPE-4CO2Na is cell-permeable but the accumulation amount in
cells in a short time (10 min) is very small. To facilitate the cell perme-
ability of the probe, its acidic form TPE-4CO2H was used in a mixed sol-
vent. Fig. 5 shows laser scanning confocal microscopy images of
can eliminate the interference from Al3+ during the detection of Pb2+
.
Fig. 4B illustrates the outstanding masking effect of NaBF4 to Al3+ and
a good PL response to Pb2+ in the presence of NaBF4 and Al3+. With
the assistance of masking reagents GSH and NaBF4, discriminative detec-
tion of Al3+ and Pb2+ can be readily achieved using TPE-4-CO2Na. Fig. 4C
shows a gradual PL enhancement of TPE-4CO2Na as the continuous addi-
tion of Al3+ in the range of 0.0–650.0 μM, and a good linear relationship
between PL intensity and Al3+ concentration in the range of 20.0–400.0
μM is obtained (Fig. S16). The detection limit according to this calibra-
tion curve was estimated to 0.7 μM. According to Table S1, the detection
limit is inferior to those of several preceding AIEgen-based methods
Fig. 5. Laser scanning confocal microscopy images in seedling roots of Arabidopsis thaliana under different conditions: (a, d) mock control; (b, e) incubated with TPE-4CO2H (0.5 mM) for
30 s; (c) incubated with TPE-4CO2H (0.5 mM) and Al3+ (0.5 mM) for 30 s; (f) incubated with TPE-4CO2H (0.5 mM) and Pb2+ (0.5 mM) for 30s.

P. Xu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117335
7
Fig. 6. Laser scanning confocal microscopy images of seedling roots of Arabidopsis thaliana radiated with different time lengths. (a, d) 1 min, (b, e) 3 min, (c, f) 5 min.
different groups of seedling roots. Almost no fluorescence signal was re-
corded for mock control under the given set of conditions, and only very
weak fluorescence was observed for root cells of Arabidopsis seedlings
treated with TPE-4CO2H, indicating that TPE-4CO2H will not aggregate
under the physiological conditions inside living cells. Continuous incu-
bation of the seedlings with a given concentration of Al3+ for a short
time (10 min) resulted in a bright green fluorescence, and almost all
the cells were clearly labeled with intense green color. However, the in-
troduction of comparable amount of Pb2+ to Al3+ did not lead to the
generation of intense emission signals. These results show that TPE-
4CO2H is capable of discriminatively imaging Al3+ over Pb2+ due to
the existence of abundant glutathione inside living cells. The
photostability of TPE-4CO2H was further evaluated after bound with
aluminum ions. Fig. 6 shows fluorescence intensity changes as the elon-
gation of irradiation time from 1 to 5 min. It is found that there is no ap-
parent decline in fluorescence intensity and no appreciable
photobleaching occurs in a given time course by comparing the laser
scanning confocal microscopy images, indicating that the probe has
good photostability and can be suited for long-term imaging.
that the acidic form of the probe is cell-permeable and is capable of se-
lectively imaging aluminum ions in living cells. The excellent
photostability of fluorescent aggregates consisting of the probe and alu-
minum ion illustrates that the designed probe is suited for long-term
imaging of ions without appreciable photobleaching.
Acknowledgements
We gratefully acknowledge the financial support from the National
Natural Science Foundation of China (Grant Nos. 21675143, 21775139
and 21775138), and Natural Science Foundation of Zhejiang Province
(Grant Nos. LR18B050001 and LY17B050003).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2019.117335.
References
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In summary, a water-soluble molecular probe that is capable of dis-
criminative detection of aluminum ion and lead ion based on ion-
triggered aggregation-induced emission is reported. The molecular
probe was composed of a tetraphenylethylene unit and four carboxylic
acid groups, and the introduction of four carboxylic acid groups facili-
tates the water-solubility of designed probe to avoid the self-
precipitation in aqueous solution. The probe shows specific response
to aluminum ion and lead ion in a fluorescence turn-on manner due
to high binding affinities of four carboxylic acid groups to aluminum
ion and lead ion. With the assistance of different masking reagents,
this probe can be used to discriminate aluminum ion and lead ion in
qualitative and quantitative way. Live-cell imaging results demonstrate
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