A new off-on fluorescent probe for Al3+ in aqueous solution based on rhodamine B and its application to bioimaging

Article abstract of DOI:10.1016/j.saa.2015.07.062

In this paper, a new fluorescent probe has been synthesized and applied as off-on sensor for the detection of Al³⁺ with a high sensitivity and excellent selectivity in aqueous media. The sensor was easily prepared by one step reaction between rhodamine B hydrazide and pyridoxal hydrochloride named RBP. The structure of the sensor has been characterized by nuclear magnetic resonance and electron spray ionization-mass spectrometry. The fluorescence intensity and absorbance for the sensor showed a good linearity with the concentration of Al³⁺ in the range of 0-12.5 μM and 8-44 μM, respectively, with detection limits of 0.23 μM and 1.90 μM. The sensor RBP was preliminarily applied to the determination of Al³⁺ in water samples from the lake of Henan University and tap water with satisfying results. Moreover, it can be used as a bioimaging reagent for imaging of Al³⁺ in living cells.

Full text of DOI:10.1016/j.saa.2015.07.062

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 70–76
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A new ‘‘off–on’’ fluorescent probe for Al3+ in aqueous solution based on
rhodamine B and its application to bioimaging
⇑
⇑
Qi Huang, Qingyou Zhang , Enze Wang, Yanmei Zhou , Han Qiao, Lanfang Pang, Fang Yu
Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan 475004, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
The sensor was designed based on
spirolactam ring-opening reaction.
The sensor showed high selectivity
for Al3+ in aqueous solution.
The sensor can be applied in water
samples and imaging of Al3+ in living
cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2015
Received in revised form 31 May 2015
Accepted 12 July 2015
Available online 14 July 2015
In this paper, a new fluorescent probe has been synthesized and applied as ‘‘off–on’’ sensor for the detec-
tion of Al3+ with a high sensitivity and excellent selectivity in aqueous media. The sensor was easily pre-
pared by one step reaction between rhodamine B hydrazide and pyridoxal hydrochloride named RBP. The
structure of the sensor has been characterized by nuclear magnetic resonance and electron spray
ionization-mass spectrometry. The fluorescence intensity and absorbance for the sensor showed a good
linearity with the concentration of Al3+ in the range of 0–12.5
tion limits of 0.23 M and 1.90
lM and 8–44
lM, respectively, with detec-
Keywords:
l
l
M. The sensor RBP was preliminarily applied to the determination of Al3+
Rhodamine B
Fluorescent sensor
Al3+
Aqueous solution
Cellular imaging
in water samples from the lake of Henan University and tap water with satisfying results. Moreover, it
can be used as a bioimaging reagent for imaging of Al3+ in living cells.
Ó 2015 Published by Elsevier B.V.
1. Introduction
importance to develop efficient methods for the detection of Al3+
in environmental and biological samples. At present, various meth-
Aluminum is the most abundant metal element in the earth’s
crust that has been widely used in cooking utensils,
aluminum-based pharmaceuticals and food additives [1,2].
ods have been employed for the treatment of aluminum, including
electrochemical detection [6], HPLC-coupled atomic spectra [7]
and mass spectrometry [8]. Although these methods have the char-
acteristics of the fast measurement and good sensitivity, they
require complicated sample-pretreatment procedures [9].
Compared with these methods, colorimetric and fluorimetric
methods have not only the advantages of operational simplicity,
high selectivity, sensitivity and rapidity, but also nondestructive.
In the past decades, some chemists and environmentalists has
been drawn to study selective and sensitive fluorescent sensors
However, aluminum is
a known neurotoxic element which
involved many neurodegenerative diseases such as Parkinson’s
disease and Alzheimer’s disease [3–5]. Thus, it is of great
⇑
Corresponding authors.
E-mail addresses: zhqingyou@henu.edu.cn (Q. Zhang), zhouyanmei@henu.
edu.cn (Y. Zhou).
http://dx.doi.org/10.1016/j.saa.2015.07.062
1386-1425/Ó 2015 Published by Elsevier B.V.

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71
for biological and environmental applications, especially in molec-
ular biology [10]. Up to now, a lot of scientific and technological
achievements have been made in the preparation and characteriza-
tion of fluorescent probes to detect metal ions in environmental
samples and imaging of them in living cells [11–18]. Despite these
discoveries, there are comparatively few compounds that target
Al3+ with high affinity and specificity.
As we all know, rhodamine is an ideal platforms for develop-
ment of fluorescent probe owing to its excellent photophysical
properties [19,20]. Specifically, it is based on spirolactam (nonflu-
orescent) to ring-opened amide (fluorescent) process utilized for
the detection of metal ions [21]. Based on this mechanism, our
ambition was to design and synthesize a new fluorescent probe
for detection of Al3+in aqueous media.
by one-step condensation between RBH and pyridoxal hydrochlo-
ride in absolute ethanol as shown in Scheme 1. In a 100 mL flask,
RBH (2 mmol, 0.92 g) was dissolved in 30 ml of absolute ethanol,
and then pyridoxal hydrochloride (4 mmol, 0.4 g) was added to
the reaction mixture. The mixture was stirred and refluxed for
10 h. After cooling to room temperature and vacuum concentrat-
ing, the residue was purified by recrystallization with ethanol to
afford compound RBP (0.35 g) as brownish-red solid. Mass spec-
trometry (ESI): m/z, calcd: 605.30, found: 606.5 ([RBP + H]+),
628.6 ([RBP + Na]+), 644.6 ([RBP + K]+). 1H NMR (400 MHz, CDCl3),
d (ppm): 8.75 (1H, s), 8.03 (1H, m), 7.82 (1H, s), 7.54 (2H, m),
7.15 (1H, d, J = 6.9 Hz), 6.51 (2H, d, J = 8.8 Hz), 6.37 (2H, d,
J = 2.4 Hz), 6.27 (2H, dd, J = 8.9 Hz, 2.6 Hz), 4.53 (2H, s), 3.32 (8H,
q), 2.45 (3H, s), 1.16 (12H, t, J = 7.1 Hz). 13C NMR (400 MHz,
Herein, we introduce a novel and simple method to prepare
rhodamine B-based chemosensor by one step reaction. The sensor
RBP showed ‘‘off–on’’ type fluorogenic and chromogenic behavior
toward Al3+ over other interfering metal ions. Furthermore, the
sensor RBP was successfully applied in detection of Al3+ in tap
water, lake water and imaging of Al3+ in living cells.
DMSO-d6) d (ppm): 164.27, 153.09, 151.90, 150.45, 149.21,
147.81, 145.73, 138.88, 135.16, 132.69, 129.62, 128.18, 124.52,
123.79, 120.31, 108.94, 104.06, 97.91, 65.72, 59.00, 49.05, 44.18,
37.08, 19.23, 12.86.
2.4. Cell culture
The living PC12 cells were provided by Medical College of
Henan University which was seeded in glass bottom culture dishes
and grew in DMEM supplemented with 2.5% Fetal Bovine Serum
and 15% horse serum at 37 °C in 5% CO2 atmosphere.
2. Experimental
2.1. Apparatus
A Hitachi F-7000 FL spectrofluorometer was used for fluores-
cence measurements. The absorption spectra were recorded with
a U-4100 Spectrophotometer. An Olympus Zeiss 710 laser scanning
confocal microscopy was used for fluorescence image of cells. 1H
and 13C NMR spectra were measured on a Bruker DMX-300 spec-
trometer at 400 MHz in CDCl3 and DMSO-d6, respectively. The
MS spectra were performed on Bruker ESQUIRE HPLC-MS AB
4000Q.
3. Results and discussion
3.1. Absorption and fluorescence response of RBP to different metal
ions
The UV/vis titration absorption spectra of RBP (10 lM) were
first explored in Tris–HCl (pH 7.2) aqueous buffer solution in the
presence of 2 equivalent of different metal ions and the results
were depicted in Fig. 1. A solution of RBP in Tris–HCl (pH 7.2) aque-
ous buffer solution is colorless. The sensor RBP exhibited very weak
absorption in the visible range. Upon binding of metal ions (Ag+,
2.2. Reagents
Rhodamine B was purchased from Beijing chemical plant,
Hydrazine hydrate was obtained from Tianjin reagent plant, pyri-
doxal hydrochloride was purchased from Aladdin. Tris–HCl aque-
ous buffer solutions of different pH were prepared using
0.2 mol/L Tris(hydroxymethyl)aminomethane (Tris) and proper
amount of HCl under adjustment by a pH meter. The solution of
metal ions was prepared from their nitrate salts or chloride salts
of analytical grade. Unless otherwise noted, the chemicals were
used as received without further purification. Distilled water was
used throughout.
Pb2+, Na+, K+, Hg2+, Cd2+, Ba2+, Zn2+, Mg2+, Cu2+, Ni2+, Ca2+, Al3+
,
Pd2+, Cr3+, Fe3+ and Pt2+), it was found that both Al3+ and Cu2+ pro-
duced a significant enhancement in absorbance and accompanied
with a clear color change from colorless to pink (Fig. 2), respec-
tively. Pd2+, Fe3+ and Pt2+ elicited a slight change of absorbance
and color, respectively.
To further explore the utility of sensor RBP as an ion-selective flu-
orescent chemosensor, the fluorogenic behavior of RBP was investi-
gated with various metal cations. Upon the addition of 2 equivalent
of each of these metal ions, the fluorescence spectra were recorded
after 30 min. As shown in Fig. 3, sensor RBP showed a very weak flu-
2.3. Synthesis of RBP
orescence. Additions of metal ions (Ag+, Pb2+, Na+, K+, Hg2+, Cd2+
,
Rhodamine B hydrazide (RBH) was synthesized according to the
literature method [22]. The chemosensor RBP was facilely prepared
Ba2+, Zn2+, Mg2+, Cu2+, Ni2+, Ca2+, Al3+, Pd2+, Cr3+, Fe3+ and Pt2+) to
the solution of RBP, only Al3+ resulted in remarkable enhancement
O
OH
O
HO
O
N
O
N
N
OH
N
N2H4.H2O
NH2
HO
Reflux
N
OH
EtOH Reflux
N
O
N
N
O
N
N
O
N
RB
RBH
RBP
Scheme 1. Synthesis of RBP.

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Q. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 70–76
Fig. 3. Fluorescent intensity of RBP (10
lM) in Tris–HCl buffer solution (pH 7.2)
Fig. 1. UV–vis absorbance of RBP (10 lM) in Tris–HCl buffer solution (pH 7.2) upon
upon addition of metal ions (20 M). kex = 530 nm.
l
addition of metal ions (20 lM).
of fluorescence intensity at 579 nm and Cu2+ elicited a slight
enhanced fluorescence intensity. Under the excitation of 530 nm,
the quantum yield of RBP is 0.09 (rhodamine B (uF = 0.97) in ethanol
was chose as a Standard). After addition of Al3+, it exhibited a strong
emission with a fluorescence quantum yield of 0.27 [23–25]. The
results showed that RBP was a good fluorescent probe toward Al3+
over other metal ions and could serve as a sensitive naked eye fluo-
rescent probe for Al3+ in aqueous solution.
3.2. Effect of pH and response time
For practical application, the fluorescence spectra response of
RBP (10 l lM) in differ-
M) in the absence and presence of Al3+ (20
ent pH values were evaluated to find a suitable pH span in which
RBP can selectively detect Al3+ efficiently. As shown in Fig. 4, own-
ing to H+ induced spirocyclic opening, sensor RBP in Tris–HCl solu-
tion showed minor enhanced fluorescence intensity between pH
3.1 and 5.1 at a 530 nm excitation wavelength. However, there
was not enhanced fluorescence intensity and obvious characteris-
tic color of rhodamine at approximate neutral and alkalinity.
After addition of Al3+, there was an apparent fluorescence enhance-
ment between pH 5.9 and 8.5, which may be attributed to the Al3+
induced formation of rhodamine ring-opened form. In this paper,
we chose pH 7.2 to mimic physiological conditions.
Fig. 4. Fluorescence intensity of RBP (10
lM) in the absence ( ) and presence (j)
of Al3+ (20
l
M) in different pH buffer solution. kex = 530 nm.
1400
1200
1000
800
The response time of the reaction system was investigated as
shown in Fig. 5. RBP exhibited no visible variations in the absence
of Al3+, which suggested that RBP was stable in the assay condi-
tions. However, upon addition of 20 l
M Al3+, the fluorescent inten-
600
sity of RBP was enhanced to a constant value after 25 min
interaction. Therefore, a 30 min reaction time was selected in order
to make the metal ions chelate with the sensor sufficiently.
400
200
3.3. Linearity
0
20
40
60
80
100
120
Time (min)
Under the optimized condition, absorption and emission titra-
tion experiments were utilized to investigate the interaction
Fig. 5. Time course of compound RBP (10
lM) in the absence ( ) and presence (j)
M) in Tris–HCl buffer solution (pH 7.2). kex = 530 nm.
of Al3+ (20
l
between RBP (20 l
M) and Al3+. As shown in Fig. 6, with increasing
Fig. 2. Color of RBP (10
13: Al3+; 14: Pd2+; 15: Cr3+; 16: Fe3+; 17: Pt2+
lM) and RBP with different metal ions (20 l ;
M), 1: Ag+; 2: Pb2+; 3: Na+; 4: K+; 5: Hg2+; 6: Cd2+; 7: Ba2+; 8: Zn2+; 9: Mg2+; 10: Cu2+; 11: Ni2+; 12: Ca2+
.

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73
Al3+ ion concentrations, the absorption band at 557 nm increased
gradually and had a linear response relationship of RBP toward
Al3+ in the range of 8–44
l
M. Based on the definition by IUPAC
(CDL = 3Sb/m), the limit of detection for Al3+ is up to 1.90
l
M.
Subsequently, the ability of RBP as a fluorescent probe for Al3+
detection by fluorescence response was carried out under the same
condition at an excitation wavelength of 530 nm. As shown in
Fig. 7, a significant increase of the fluorescence intensity at
579 nm was observed with increasing Al3+ concentrations over
the range of 0–12.5 lM. The regression equation is
Y = 199.54 + 119.62X (R = 0.9973). Based on the definition by
IUPAC (CDL = 3Sb/m), the limit of detection for Al3+ is up to 0.23
lM.
3.4. Proposed binding mode
Fig. 8. Job’s plot of the complex formed by [Al3+]/([RBP] + [Al3+]). The total
concentration of RBP and Al3+ was 20
lM. The wavelength of absorbance is 557 nm.
The binding stoichiometry of the RBP-Al3+ complex was studied
based on literature method [26]. Job’s plot experiment was estab-
lished from the absorption spectra of RBP and Al3+under a constant
total concentration. A plot of absorption versus the molecular frac-
tion of [Al3+]/([RBP] + [Al3+]) was provided in Fig. 8. It established
that the maximum absorption was about 0.33 indicating the 2:1
stoichiometry between RBP and Al3+ in the inclusion complex.
To further verify the complexation behavior of RBM with Al3+
,
the ESI-MS experiment was carried out. As shown in Fig. 9, the
peak at m/z = 606.5 corresponded to [RBP + H]+, and the peak at
m/z = 619.2, 649.0 and 1236.1 corresponded to [2(RBP-H) +
Al + 2H]2+
,
[2(RBP-H) + Al + K + Na]2+ and [2(RBP-H) + Al + H]+,
Fig. 6. (a) Absorbance spectra of reaction solution of RBP (20
at 557 nm as a function of Al3+ concentration. All error bars were obtained through the detection of three parallel samples.
l
M) in Tris–HCl buffer solution (pH 7.2) with different concentrations of Al3+. (b) The corresponding absorbance
Fig. 7. (a) Fluorescent spectra of reaction solution of RBP (20 l
M) in Tris–HCl buffer solution (pH 7.2) with different concentrations of Al3+. Excitation wavelength was
530 nm. (b) The corresponding fluorescence intensity at 579 nm as a function of Al3+ concentration. All error bars were obtained through the detection of three parallel
samples.