A facile Al(iii)-specific fluorescence probe and its application in biological systems

Article abstract of DOI:10.1039/c6ra17623b

A facile fluorescence receptor was easily synthesized by an one-step reaction of 3,4-dihydroxybenzaldehyde and hydrazinecarbothioamide. The receptor as a fluorescence probe was used to recognize metal ions by UV-vis and fluorescence techniques. The data o

Full text of DOI:10.1039/c6ra17623b

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A facile Al(III)-specific fluorescence probe and its
application in biological systems†
Cite this: RSC Adv., 2016, 6, 77291
a
b
b
b
c
d
*
*
Haiying Lei, Haipeng Diao, Wen Liu, Jun Xie, Zhijun Wang and Liheng Feng
A
facile fluorescence receptor was easily synthesized by an one-step reaction of 3,4-
dihydroxybenzaldehyde and hydrazinecarbothioamide. The receptor as a fluorescence probe was used
to recognize metal ions by UV-vis and fluorescence techniques. The data of UV-vis and fluorescence
spectra display the high sensitivity and selectivity of the receptor for Al(III) ions. When introducing Al(III)
ions, the fluorescence of the probe exhibits an obvious enhancement because of inhibiting the
photoinduced electron transfer (PET) process. The certain bonding mode of the receptor with Al(III) was
1
verified by H NMR, HRMS-ESI and Job-plot data. The prospective fluorescence “Off–On” signal for Al(III)
Received 10th July 2016
Accepted 8th August 2016
ions including the color change of the receptor solution was observed. The detection limit of the
receptor for Al(III) may reach 10ꢀ7 mol Lꢀ1. Noticeably, the probe as a fluorescence “Off–On” switch can
detect the trace level of Al(III) in living cells and mouse organs. The work provides a strategy for design
simple fluorescence probes for Al(III) and applications in biological systems.
DOI: 10.1039/c6ra17623b
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developed effective aluminum probes with high selectivity and
sensitivity in the environmental and biological samples are very
Introduction
urgent for chemist, biologist and medical scientist. In recent
years, carbon dots and graphene quantum dots have also been
widely used as uorescence probes for the detection of transi-
tion metal ions because of their high uorescence brightness
and sensitivity.28–31 Compared with transition metal ions, it is
challenging to develop excellent uorescence probes for Al3+
because of its poor coordination ability and spectroscopic
characteristics.32–34 To date, among reported Al3+ uorescence
probes (such as hydrazones, coumarin, calixarene and hydrox-
yavone, etc.), Schiff base and its derivants as uorescence
receptors are the most effective and prosperous because of their
easy preparation and abundant bonding sites (N and O
atoms).35–40 Additionally, due to the isomerization characteristic
of C]N bond, the probes based on Schiff base always display
a uorescence “off–on” signal for analytes. Through utilizing
the feature, many Al3+ uorescence probes with Schiff base
formation have been reported in recent years.41–51 Meanwhile,
these researches mainly focused on improving the parameter
values of sensitivity and selectivity for Al3+, and a few applica-
tions in biological systems. Surprisingly, it is negligible that the
C]N bond also is a poor electron group which possesses the
strong withdrawing capacity. So, it is interest for designing
a uorescence probe with the withdrawing electron unit (C]N
bond of Schiff base) and donating electron groups (such as
hydroxyl or amino) because the probe can use to recognize the
specic ions based on photoinduced electron transfer (PET)
mechanism. Furthermore, considering the human health
problem, it is signicant to develop a facile uorescence probe
to recognize and detect trace level of Al3+ in biological systems.
Because of its operational simplicity, low cost, instantaneous
response and direct visual perception, the uorescence technique
has proved to be a very useful tool for sensing and monitoring
metal ions, anions and molecules in an abiotic or biotic
systems.1–10 A typical uorescence probe is comprised of a substrate
(as the recognition site), a chromophore (the optical signal source)
and a connection bridge (translating the recognition event into
a uorescence signal).11–15 In order to get ideal probes, a well
designed uorescence receptor is very important, and the receptor
needs to possess strong affinity towards the relevant targets.16–19
Aluminum has been widely used in packing materials, clin-
ical drugs, food additives and water purication because of its
rich feature in the earth (the third most prevalent metallic
element).20–24 However, many health hazards have produced in
recent years because of the improper use of aluminum.25,26
Under the case, World Health Organization (WHO) drew up the
safety dosage of aluminum. And, the organization also revealed
aluminum as a source of food pollution and limited the content
of aluminum in drinking water to 7.41 mM.27 Therefore,
aDepartment of Biological Science and Technology, Changzhi University, Changzhi,
046011, P. R. China
bSchool of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, P. R.
China
cDepartment of Chemistry, Changzhi University, Changzhi, 046011, P. R. China.
E-mail: czxywzj@163.com
dSchool of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, 030006,
P. R. China. E-mail: lhfeng@sxu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra17623b
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Herein, we report a simple receptor as a uorescence probe of receptor 1 with various metal ions, respectively. The uo-
to recognize Al3+ ions. The receptor ((E)-2-(3,4-dihydroxy- rescence spectra of receptor 1 in the present of metal ions were
benzylidene)hydrazine-1-carbothioamide, receptor 1, Fig. 3) shown in Fig. 2. From Fig. 2, the receptor 1 has a weak uo-
was comprised of withdrawing electron unit (C]N bond), rescence emission at 410 nm because of the intramolecular
donating electron groups (hydroxyl) and a connection bridge photoinduced electron transfer from hydroxyl groups to C]N
(benzene ring). As expected, the probe exhibits a uorescence unit of Schiff base. An obvious enhancement of uorescence
“turn-on” response only for Al3+ ions because of inhibiting intensity of receptor 1 is observed by only introducing Al3+
.
photoinduced electron transfer (PET) process from hydroxyl While introducing Hg2+, Cu2+, Fe3+ and Pb2+ metal ions, the
groups to C]N bond. Moreover, the detections of the probe for uorescence of receptor 1 is quenched, respectively. The uo-
Al3+ in vitro cells and mouse organs also were investigated.
rescence of receptor 1 has not an obvious change in the present
of other metal ions, respectively. Based on the experimental
facts, we believe the receptor can detect Al3+ as a high selectivity
probe. Meanwhile, the sensitivity of receptor 1 for Al3+ is also
studied by the titration method at 445 nm (Fig. S2†). With the
increase of Al3+, the emission peak exhibits a red-shied from
410 nm to 445 nm with the gradual enhancement of uores-
cence intensity (Fig. S2†). The detection limit of receptor 1 for
Al3+ is up to 10ꢀ7 mol Lꢀ1, which is low enough as a uorescence
probe to detect Al3+ in environmental and biological samples.31
Notably, the plot of relative intensity to the concentration of Al3+
ions possesses a good linear relation and reproducibility
(Fig. S2†). The linear independent constant (R2) is up to 0.9945
within the range of 10ꢀ7 to 10ꢀ5 mol Lꢀ1 for Al3+. Compared
with electrochemical technique for detection Al3+, the advan-
tages of the uorescence probe lie in its low cost, fast analysis
and wide application.52
Fluorescence spectra of the probe in different pH solutions
also were investigated by the titration method (Fig. S3†). In pH <
5.0 media, the emission peak of receptor 1 at 405 nm has no
obvious changes because of the protonation of N and O atoms
weakening the conjugated extend, which results in the blue-
shied of uorescence spectra.53 The wavelength and intensity
of the probe exhibit a red-shied and enhancement form pH 5.0
to pH 7.0. And, the uorescence of the probe with an
outstanding reduction in pH > 8.0 media may be due to the
dissociation of the hydroxyl and amino protons.54 The result
indicates that the pH value has an obvious effect for the uo-
rescence of the probe. The acid (pH < 5.0) or alkali (pH > 8.0)
Results and discussion
UV-vis studies of receptor 1 and metal ions
The interactions of receptor 1 with various metal ions (Zn2+,
Pb2+, Ni2+, Na+, Mn2+, Mg2+, K+, Hg2+, Fe2+, Fe3+, Cu2+, Cr3+, Co2+,
Cd2+, Ca2+, Ag+ and Al3+) were studied by UV-vis absorption
spectra in mixed media (H2O/DMSO ¼ 2/3, v/v), respectively. As
shown in Fig. 1, the absorption peak of receptor 1 is at 335 nm.
The absorption spectra of receptor 1 exhibit an obvious blue-
shied and enhancement in intensity with the addition of
Cu2+, Fe3+, Hg2+ and Pb2+, respectively. Different from the above
four transition metal ions, the absorption peak of receptor 1
displays a red-shied only in the present of Al3+. Meanwhile, the
introduction of other metal ions does not induce obvious change
of the absorption peak of receptor 1 in wavelength or intensity
aspects. These results show that the receptor 1 has a unique
response and high selectivity for Al3+. The change processes of
the absorption spectra with the increase of Al3+ were listed in
Fig. S1.† From Fig. S1,† we can nd that the absorption peak of
receptor 1 displays an obvious change from 350 nm to 360 nm,
and the isosbestic point is 352 nm, which indicates the forma-
tion of a complex between receptor 1 and Al3+.
Fluorescence studies of receptor 1 and metal ions
In order to conrm the selectivity of receptor 1 for Al3+, the
uorescence technique was used to investigate the interactions
Fig. 1 The UV-vis absorption spectra of receptor 1 and receptor 1 (1.0 Fig. 2 The fluorescence spectra of receptor 1 (5.0 ꢁ 10ꢀ6 mol Lꢀ1) and
ꢁ 10ꢀ5 mol Lꢀ1) with various metal ions (5.0 ꢁ 10ꢀ4 mol Lꢀ1) in mixed introducing various metal ions (3.0 ꢁ 10ꢀ5 mol Lꢀ1) to receptor 1 in
media (H2O/DMSO ¼ 2/3, v/v), respectively.
mixed medium (H2O/DMSO ¼ 2/3, v/v), respectively.
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media can both inhibit the uorescence of the probe. Although the HepG-2 cells were cultured with receptor 1 at 37 ꢂC for 1 h or
the uorescence of the probe in pH ¼ 7.0 medium obtains 3 h, and then, the ve equivalents of Al3+ were added to the
a certain recovery, it is lower than that in present of Al3+, which mixture and sequentially cultured the cells for 1 h. The uo-
indicates the detection of the probe for Al3+ has no apparent rescence imaging was taken, where Lyso tracker red was used as
effect during pH 5.0–8.0 range.
a cytoplasm located dye. As shown in Fig. 4, the blue uores-
cence of receptor 1 can be observed only in the present of Al3+
,
and the staining region has well overlap with the cytoplasm
located dye (Lyso tracker red). Compared with receptor 2, the
The sensing mechanism and bonding mode
For uorescence probes, the response signals for analytes
mainly included the following mechanisms, such as photo
induced electron transfer (PET), intramolecular charge transfer
(ICT), chelation enhanced uorescence (CHEF) and twisted
intra-molecular/intermediate charge transfer (TICT), etc. In this
work, the uorescence of the probe is inhibited because of
photoinduced electron transfer action from rich electronic
group (hydroxyl group) to lack electronic body (C]N bond of
Schiff base). When introducing Al3+, the process of PET is
destroyed by the connection of receptor 1 and Al3+. Accordingly,
the “turn-on” response signal of the probe for Al3+ is observed
with the color change of the solutions by naked eyes (Fig. 3).
receptor 1 in live cells can be illumed by introducing Al3+
.
While, due to shorter incubation time of receptor 1 with HepG-2
cells (1 h), the uorescence imaging is not bright (Fig. S7†).
These results show that receptor 1 can detect Al3+ in living cells
as a biocompatible and effective uorescence probe.
Fluorescence imaging in mouse organs of receptor 1 for Al3+
In order to further develop the application in biological eld,
uorescence imaging of the probe for Al3+ was performed in
mouse organs (Fig. 5). As shown from Fig. 5, typical ex vivo
biophotonic images of mouse main organs (heart, liver, spleen,
lung, kidney and brain) at 1 h post injection of receptor 1/Al3+
were investigated. In this experiment, propidium iodide (PI) is
used as a located dye for these organs. The blue uorescence is
observed at various organs of mouse by introducing Al3+ to
these organs with receptor 1, which indicates receptor 1 as
a suitable “off–on” probe may apply to detect Al3+ in vivo.
In order to explore the action mode of receptor 1 with Al3+
,
other two receptors (2 and 3) were prepared and applied to
detect the Al3+ in Fig. S4.† As can be seen from Fig. S4,† we can
obtain other two receptors have similar structures with receptor
1, but the two receptors have all no uorescence changes in the
present of Al3+, respectively. Combining with the 1H NMR
titration data (Fig. S5†), only a change of chemical shi was the
hydroxyl groups, which indicates oxygen atoms of hydroxyl
participate in the complexation with Al3+. Next, the specic
mode of receptor 1 with Al3+ was veried by HRMS-ESI data
(Fig. 3). Based on these cases, we believe the bonding ratio of
receptor 1 with Al3+ is 1 : 1 (mol) mode, which also was veried
by the Job-plot experiment (Fig. S6†). In the Job-plot testing, the
uorescence intensity of receptor 1 with Al3+ (5 : 5, mol) was
highest in different ratios.
Live subject statement
These authors state that all experiments were performed in
compliance with the relevant laws and institutional guidelines
(Animal Ethical Committee, Shanxi Medical University) and this
work has been approved by the IAEC (Institutional Animal
Ethical Committee) constituted as per the Rules and
Fluorescence imaging in live cells of receptor 1 for Al3+
The interaction of receptor 1 with Al3+ in live cells was investi-
gated by confocal laser scanning microscope (CLSM). Firstly,
Fig. 4 Fluorescence imaging of HepG-2 cells with receptor 1, 2 and
Al3+. (a) is bright image, (b) is fluorescence image of cytoplasm located
dye (Lyso tracker red), (c) is fluorescence image of receptors or
Fig. 3 The sensing mechanism of the probe for Al3+, the bonding receptors/Al3+, (d) is merged image of located dye and receptors or
mode of receptor 1 with Al3+ and HRMS-ESI (negative) graphs, and the receptors/Al3+, (e) is merged bright image and fluorescence image of
color change of receptor 1 solution without or with Al3+
.
located dye and receptors or receptors/Al3+
.
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with a Hitachi F-4600 uorescence spectrophotometer, and the
excitation and emission slit widths were both 5.0 nm. UV-vis
spectra were measured with a Hitachi 5300 absorption spec-
trophotometer. Confocal laser scanning microscopy (CLSM)
imaging was taken on a confocal laser scanning biological
microscope (FV1000-IX81, Olympus, Japan).
Procedures for synthesis of receptors
0.46 g (5.0 mmol) hydrazinecarbothioamide was added to
a suspension of 0.69 g (5.0 mmol) 3,4-dihydroxybenzaldehyde in
30 mL ethanol, and the reaction mixture was stirred at 80 ꢂC for
5 h. Aer ltrating the insoluble precipitate and removing
a mass of ethanol, the residue was precipitated with dichloro-
methane (50 mL) and the precipitate was collected by centri-
fugation. The crude product was puried by precipitation for
twice from ethanol to dichloromethane. The nal product
(receptor 1) as solid was obtained through centrifugation and
drying under vacuum. Receptor 2 and 3 were prepared accord-
ing to the similar method.
Fig. 5 Fluorescence images of major organs, including heart, liver,
spleen, lung, kidney and brain collected from the control untreated
mouse and the injected (Al3+ or receptor 1, 5.0 ꢁ 10ꢀ5 mol Lꢀ1) mice at
1 h post-injection. No noticeable abnormality or lesion was observed
in organs of mouse.
1
Receptor 1 (deep yellow powder, yield 43.2%). H NMR (d-
DMSO, 400 MHz) d 11.20 (s, 1H), 9.46 (s, 1H), 8.98 (s, 1H), 8.03
(s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.18 (s, 1H), 7.02 (d, 1H), 6.76
(s, 1H); 13C NMR (d-DMSO, 100 MHz) d 177.18, 148.00, 145.40,
144.62, 125.86, 120.79, 114.90, 112.75; element analysis for
C8H9N3O2S (mol. wt: 211.04) calcd C, 45.49; H, 4.29; N, 19.89; O,
15.15; found: C, 45.52; H, 4.33; N, 19.95; O, 15.23; HRMS-ESI
(positive) for C8H9N3O2S (m/z) 211.91026 [M + 1].
Regulations of Ministry of Animal Husbandry, Government of
China. The authors also state that informed consent was ob-
tained for any experimentation with human subjects and
Animal Ethical Committee, Shanxi Medical University.
Conclusions
Receptor 2 (light yellow powder, yield 45.7%). 1H NMR (d-
DMSO, 400 MHz) d 11.22 (s, 1H), 9.45 (s, 1H), 9.02 (s, 1H), 8.19
(s, 1H), 8.17 (s, 1H), 8.11 (s, 1H), 7.38 (d, 1H), 6.36 (d, 1H), 6.31
(s, 1H); 13C NMR (d-DMSO, 100 MHz) d 177.19, 161.12, 158.89,
145.38, 130.39, 110.98, 107.71, 102.14; element analysis for
C8H9N3O2S (mol. wt: 211.04) calcd C, 45.49; H, 4.29; N, 19.89; O,
15.15; found: C, 45.55; H, 4.32; N, 19.81; O, 15.24; HRMS-ESI
(positive) for C8H9N3O2S (m/z) 211.94012 [M + 1], 233.92894
[M + Na].
In summary, we develop a simple uorescence “off–on” probe
for Al3+ with high selectivity and sensitivity. The probe was
comprised of pyrocatechol and thiosemicarbazide and easily
synthesized by a one step. Due to destroying the PET action by
introducing Al3+, the uorescence of the probe exhibits an
outstanding increase. The bonding mode (1/1, mol) of the
receptor with Al3+ was veried by HRMS-ESI and Job plot data.
The probe with good biocompatible and high specic for Al3+
can detect Al3+ ions in cells and organs systems. The contribu-
tion of the work lies in not only providing a new platform for
applications of uorescence probes of Al3+, but also proposing
a strategy for designing and preparing easy Schiff base probes.
1
Receptor 3 (white powder, yield 41.8%). H NMR (d-DMSO,
400 MHz) d 11.15 (s, 1H), 9.44 (s, 1H), 8.05 (s, 1H), 7.90 (s, 1H),
7.82 (s, 1H), 7.22–7.16 (m, 3H), 6.84 (t, 1H); 13C NMR (d-DMSO,
100 MHz) d 178.52, 157.53, 143.95, 135.30, 129.44, 118.90,
117.17, 112.94; element analysis for C8H9N3OS (mol. wt: 195.05)
calcd C, 49.22; H, 4.65; N, 21.52; O, 8.19; found: C, 48.95; H,
4.68; N, 22.32; O, 8.41; HRMS-ESI (positive) for C8H9N3OS (m/z)
196.22038 [M + 1].
Experimental
Materials and characterization
Unless otherwise stated, all chemical reagents were obtained
from commercial suppliers and used without further purica-
tion. Hydrazinecarbothioamide, benzaldehyde with different
Fluorescence and UV-vis measurements
hydroxyl groups were purchased from Aldrich (Steinheim, Ger- All used water was redistilled water. All these receptors were
many). All metal ions were nitrates and provided by Alfa Aesar dissolved in DMSO as the stock solutions (1.0 ꢁ 10ꢀ2 mol Lꢀ1).
(Tianjin, China). 1H NMR and 13C NMR were measured on The working solutions were prepared by diluting method with
a Bruker ARX400 spectrometer with chemical shis reported as mixed media (H2O/DMSO ¼ 2/3, v/v). The working solutions
ppm (TMS as an internal standard). Elemental analyses were were placed in a quartz cuvette with 1 cm path. The total volume
performed on a Vario EL elemental analysis instrument (Ele- of working solutions is 2.0 mL. The UV-vis and uorescence
mentar Co.). High-resolution mass spectra (HRMS) were measurements used titration experiments and the volume
acquired with an Agilent 6510 Q-TOF LC/MS instrument (Agi- added did not exceed 3% of the total. Aer the mixture solution
lent Technologies, Palo Alto, CA) equipped with an electrospray was shaken for 30 s, the new spectra were measured. All of the
ionization (ESI) source. Fluorescence spectra were acquired experiments were performed at room temperature.
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Cells and organs imaging of mouse
Human liver carcinoma cells (HepG-2) were cultured in DMEM
medium containing 10% FBS routinely under a humidied
atmosphere containing 5% CO2, and then harvested for
subculture using trypsin (0.05%, Gibco/Invitrogen) at 37 ꢂC.
HepG-2 cells were subcultured onto a 35 mm ꢁ 35 mm Petri
dish with a glass bottom, then allowed to grow for 24 h for
attachment. Aer that, 1 mL of DMEM medium containing 10%
20 mM receptor (1 or 2) was used to incubate the HepG-2 cells at
37 ꢂC for 3 h. The media were replaced and phosphate-buffered
saline (PBS, pH ¼ 7.4) was used to wash the cells thrice. And ve
equivalents of Al3+ in PBS buffer solution were added into the
ꢂ
dish and the cells were cultured at 37 C for 1 h. The medium
was replaced and phosphate-buffered saline (PBS, pH 7.4) was
used to wash the cells thrice. Then fresh medium with cyto-
plasm located dye (Lyso tracker red) was added and incubated.
Aer washing thrice with PBS, the images of the cells were
recorded on confocal laser scanning microscopy.
The mice that come from the animal laboratory of Shanxi
Medical University were rstly injected with 100 mL of 1.0 ꢁ
10ꢀ4 mol Lꢀ1 Al3+. Aer 1 h, 100 mL (1.0 ꢁ 10ꢀ4 mol Lꢀ1
)
receptor 1 was injected by tail intravenous and acted for 1 h.
Aer the anatomical and histological section, the uorescence
images of heart, liver, spleen, lung, kidney and brain organs
were recorded on confocal laser scanning microscopy.
Untreated mice were used as the control.
Acknowledgements
The work described in this paper was supported by the National
Nature Science Foundation (No. 21571116 and 21371110), the
Youth Science Foundation of Shanxi Province (No. 2014021006,
and 2016021054) and the Program for the Outstanding Inno-
vative Teams of Higher Learning Institutions of Shanxi (2013).
The authors of Haiying Lei and Haipeng Diao contributed
equally to this work.
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