Characterization of an Al3+-selective fluorescent probe based on a benzoyl hydrazine derivative and its application in cell imaging

Article abstract of DOI:10.3906/kim-1511-37

A fluorescent probe based on benzoyl hydrazine was synthesized and characterized as an Al³⁺-selective fluorescent probe. This probe showed good selectivity towards Al³⁺ compared to other common ions. Under optimized experimental cond

Full text of DOI:10.3906/kim-1511-37

Turk J Chem
Turkish Journal of Chemistry
(2016) 40: 625 – 630
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1511-37
Research Article
Characterization of an Al3+ -selective fluorescent probe based on a benzoyl
hydrazine derivative and its application in cell imaging
Yuxiang JI, Chunwei YU, Shaobai WEN, Jun ZHANG∗
Department of Environmental Sciences, School of Tropical and Laboratory Medicine, Hainan Medical College,
Haikou, P.R. China
Received: 11.11.2015
•
Accepted/Published Online: 25.01.2016
•
Final Version: 21.06.2016
Abstract: A fluorescent probe based on benzoyl hydrazine was synthesized and characterized as an Al3+ -selective
fluorescent probe. This probe showed good selectivity towards Al3+ compared to other common ions. Under optimized
experimental conditions, the probe exhibited a linear dynamic response for Al3+ from 5.0 × 10−7 to 4.5 × 10−6
M
with a detection limit of 1.3 × 10−7 M in ethanol–water solution (9:1, v:v, pH 6.8, 20 mM HEPES). Furthermore, it
was used for imaging of Al3+ in living cells with satisfying results.
Key words: Fluorescent probes, Al3+ , cell imaging
1. Introduction
Fluorescence techniques offer significant advantages over other methods for species monitoring inside living
cells because of the nondestructive character, instantaneous response, and availability of a wide range of
indicator dyes, and many biologically important species such as metal ions, anions, and amino acids have
been successfully detected by this method in vitro and in vivo.1−3 Because of the attractive electronic and
photophysical properties of metal complexes of Schiff bases, particular attention has been paid to the synthesis
and study of these compounds.4,5 In addition, Schiff base derivatives incorporating a fluorescent moiety are
appealing tools for optical sensing of metal ions.4−8 Among the metal ions, aluminum is a nonessential element
for living systems, but the ionic radius and charge of Al3+ make it a competitive inhibitor of several essential
8
elements like Mg2+ , Ca2+ , and Fe3+
.
Therefore, the detection of chelatable aluminum (Al3+) in biological
studies has attracted much attention recently.9−11 However, the lack of spectroscopic characteristics and poor
coordination ability compared to transition metals mean the detection of Al3+ has always been problematic.8
For this reason, the development of Al3+ probes is more difficult than those of other metal ions. In general,
Al3+ , being a hard acid, prefers hard donor sites like N and O in its coordination sphere. As a result, most of
the reported Al3+ probes contain mixed N and O donor sites.8,12−14 With the above-mentioned in mind, in this
work a Schiff base compound containing N and O donor sites was synthesized and successfully characterized as
an Al3+ -selective probe (Scheme 1).
∗Correspondence: jun zh1979@163.com
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O
O
CHO
HN
N
OH
NHNH2
HC
+
OH
P
Scheme 1. Synthesis route of probe P.
2. Results and discussion
2.1. Effects of pH on P and P with Al3+
The influence of pH on fluorescence was determined first. As shown in Figure 1, the emission intensities of the
free probe P can be negligible in the range pH 4–10, suggesting that probe P is stable over a wide pH range.
However, a significant fluorescence enhancement was measured upon addition of Al3+ in the pH range 4–6.8,
which is attributed to coordination of P with Al3+ . For the natural sample considered, further UV-vis and
fluorescent studies were carried out in ethanol–water solution (9:1, v:v, 20 mM HEPES, pH 6.8).
300
200
100
0
4
5
6
7
8
9
10
Figure 1. pH-dependent spectrum of P (10 µM) (•) and P (10 µM) plus Al3+ (50 µM) (■) in HEPES buffers as a
function of different pH values in ethanol–water solution (9:1, v:v, 20 mM HEPES).
2.2. UV-vis spectral response of P
Absorption spectra of P were obtained in ethanol–water solution (9:1, v:v, 20 mM HEPES, pH 6.8) as shown
in Figure 2. The addition of Al3+ to the solution of P (10 µM) caused an obvious red-shift in the UV region
(Figure 2a), and with the addition of different concentration of Al3+ , there was a regular change in the UV
spectra (Figure 2b). These results clearly suggested the binding of P with Al3+
.
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0.5
0.4
0.3
0.2
0.1
0.0
b)
a)
0.20
0.15
0.10
0.05
0.00
[Al3+]
P+Al3+
P
250
300
350
400
450
500
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Figure 2. a) The absorption spectra of P (10 µM) with Al3+ (50 µM) in ethanol–water solution (9:1, v:v, pH6.8, 20
mM HEPES); b) Absorbance of P (10 µM) with various concentrations of Al3+ (0–10 µM) in ethanol–water solution
(9:1, v:v, pH 6.8, 20 mM HEPES).
2.3. Fluorescent signaling of Al3+
For an excellent probe, high selectivity is a matter of necessity. Related metal ions, including Na+ , K+ , Ca2+
,
Cd2+ , Mg2+ , Co2+ , Zn2+ , Pb2+ , Ni2+ , Hg2+ , Ag+ , Cu2+ , Fe3+ , Al3+ , and Cr3+ , were used to evaluate
the metal ions binding properties of P by fluorescence spectroscopy (Figure 3). The results showed that the
proposed probe P has good selectivity to Al3+ , which was also confirmed by the interference experiment (Figure
S1). Upon the addition of increasing concentration of Al3+ , the intensity increased drastically, and a linear
relationship was observed to exist between the relative fluorescent intensity and the concentration of Al3+ in
the range of 5.0 × 10−7 to 4.5 × 10−6 M with a detection limit of 1.3 × 10−7 M (Figure 4).
150
Al3+
150
[Al3+]
120
120
90
90
60
60
Zn2+
30
blank and other cations
30
0
0
450
500
550
600
650
450
500
550
600
650
Wavelength
Wavelength (nm)
Figure 3. Fluorescent emission spectra of P (10 µM) to
different metal ions (50 µM) in ethanolwater solution (9:1,
v:v, pH 6.8, 20 mM HEPES).
Figure 4. Fluorescence spectra of P (10 µM) in the pres-
ence of different amounts of Al3+ (0–10 µM) in ethanol–
water solution (9:1, v:v, pH 6.8, 20 mM HEPES).
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JI et al./Turk J Chem
2.4. The proposed reaction mechanism
The method of continuous variation (Job’s method) was used to determine the stoichiometry of the P–Al3+
complex (Figure 5). As expected, the result indicated a 1:1 stoichiometry of Al3+ to P in the complex. In the
mass spectra of Al3+ –P complex (Figure S2), 351.1 corresponded to [P + Al3+ + Cl− – H+ ]+ and 387.7
corresponded to [P + Al3+ + 2Cl− ]+ , also supporting the binding mode of P with Al3+ . The association
constant K was determined from the slope to be 2.2 × 104
M
−1 , by plotting the fluorescence intensity 1/(F −F0)
against 1/[Al3+ ]. According to the results, the plausible binding mechanism of P in the present system is
schematically depicted in Scheme 2, and the enhancement of fluorescence may be caused by blocking the C=N
isomerization rather than another mechanism. A reversibility experiment was carried out and the results showed
that the reaction of Al3+ with proposed probe P was reversible (Figure S3).
100
80
60
40
20
0.0
0.2
0.4
0.6
0.8
1.0
[P]/[P+Al3+]
Figure 5. Job’s plot of P with Al3+ in ethanol–water solution (9:1, v:v, pH 6.8, 20 mM HEPES). Total concentrations
of P and Al3+ were kept at a fixed 20 µM.
H
C
H
N
H
C
H
N
O
Al3+
N
N
O
OH
HO
P
P + Al3+
Scheme 2. Proposed binding mode between P and Al3+
.
2.5. Preliminary analytical application
To further demonstrate the practical applicability of the probe P to detect Al3+ in living cells, fluorescence
images of Hl-7701 cells were recorded before and after addition of Al3+
.
The cells were supplemented with only P in the growth medium for 30 min at 37 ◦ C, which led to no
fluorescence as determined by laser scanning confocal microscopy (ex = 405 nm) (Figure 6a). In contrast, when
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loaded with 1 µM AlCl3 for 30 min, a bright fluorescence was detected (Figure 6b). These results suggested
that probe P can penetrate the cell membrane and might be used for detecting Al3+ in living cells.
Figure 6. Confocal fluorescence images in Hl-7701 cells (ex = 405 nm). (a) Cells incubated with 20 µM P in PBS
buffer for 30 min; (b) Cells incubated with 20 µM P in PBS buffer for 30 min, and then further incubated with 1 µM
Al3+ for 30 min, washed three times; (c) Brightfield image of cells shown in panel a) and b); (d) Overlay of b) and c).
3. Experimental section
3.1. Reagents and instruments
All reagents and solvents were of analytical grade and used without further purification.
UV-Vis spectra were obtained on a Hitachi U-2910 spectrophotometer. Fluorescence emission spectra
were obtained on a Hitachi 4600 spectrofluorometer. Mass (MS) spectra were recorded on a Thermo TSQ
Quantum Access Agilent 1100 system. Nuclear magnetic resonance (NMR) spectra were measured with a
Bruker AV 400 instrument and chemical shifts are given in ppm from tetramethylsilane (TMS).
3.2. Synthesis of compound P15
2-Hydroxy-1-naphthaldehyde (1.0 mmol) and benzoichydrazide (1.0 mmol) were mixed and stirred in ethanol
(30 mL) at 80 ◦ C for 4 h and then cooled to room temperature. The white precipitate so obtained was filtered
and dried under vacuum and used directly. Yields: 85.3%. MS: m/z 291.30 [M + 1]+ ; 313.22 [M + Na]+ . 1 H
NMR (δ ppm, d6 -DMSO): 12.81 (s, 1H), 12.23 (s, 1H), 9.52 (s, 1H), 8.24 (d, 1H, J = 8.5), 8.01 (d, 2H, J =
7.2), 7.95 (d, 1H, J = 9.0), 7.91 (d, 1H, J = 8.1), 7.67 (d, 1H, J = 7.4), 7.64 (d, 1H, J = 5.4), 7.63 (d, 1H,
J = 7.9), 7.59 (d, 1H, J = 7.0), 7.43 (t, 1H, J = 7.4), 7.26 (d, 1H, J = 9.0). 13 C NMR (δ ppm, d6 -DMSO):
163.42, 158.92, 147.77, 133.64, 133.58, 132.99, 132.53, 129.89, 129.55, 128.72, 128.67, 128.48, 124.44, 121.49,
119.81, 109.43 (Figures S4–S6).
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3.3. General spectroscopic methods
Metal ions and probe P were dissolved in deionized water and DMSO to obtain 1.0 mM stock solutions, respec-
tively. Before spectroscopic measurements, the solution was freshly prepared by diluting the high concentration
stock solution to the corresponding desired concentration. For all measurements, excitation and emission slit
widths were 5 nm and excitation wavelength was 405 nm.
4. Conclusions
In summary, we describe an Al3+ -selective fluorescent probe. This proposed probe has good selectivity and
sensitivity to Al3+ compared to other common ions. In addition, we have demonstrated that P can be used to
detect Al3+ in living cells. It is anticipated that the proposed probe will significantly promote studies on the
effects of Al3+ in biological systems.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (No. 81260268,
81560347) and the Colleges and Universities Scientific Research Projects of the Education Department of Hainan
Province (Hnky2015-42) and the Natural Science Foundation of Hainan Province (No. 20164164).
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Supplementary
Characterization of an Al3+ -selective fluorescent probe based on a benzoyl hydrazine
derivative and its application in cell imaging
Yuxiang JI, Chunwei YU, Shaobai WEN, Jun ZHANG
Department of Environmental Sciences, School of Tropical and Laboratory Medicine, Hainan Medical College,
Haikou, P.R. China
Figure S1. Fluorescence response of P (10 µM) to 10 µM Al3+ and to the mixture of 50 µM other metal ions with
10 µM Al3+
.
+Q1: 0.050to0.101minfromSample1(TuneSampleID)of MT20160108173138.wiff (TurboSpray)
Max. 4.1e6cps.
389.0
387.7
4.0e6
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
1.8e6
1.6e6
1.4e6
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.0
351.1
390.2
313.2
318.4
347.1
338.4
333.2
353.2
329.2
302.3
352.1
363.5
362.3
314.2
369.2
330.3
303.4
306.4
391.3
319.3
334.3
335.3
339.5
348.1
344.2
345
345.1
354.0
355.1
355
361.3
360.4
364.3
386.3
371.1
310.3
310
331.2
374.5
375
316.2
317.2
320
304.3
300
337.1
340
380.2
380
392.2
321.3
325
394.2
395 400
305
315
330
335
350
m/z, Da
360
365
370
385
390
Figure S2. ESI-MS mass spectrum of P with Al3+
.
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JI et al./Turk J Chem
Figure S3. Reversible titration response of P to Al3+ in ethanol–water solution (9:1, v:v, pH 6.8, 20 mM HEPES) (a)
P (10 µM); (b) P (10 mM) with Al3+ (50 µM); (c) P (10 µM) with Al3+ (50 µM) and then addition of EDTA (100
µM); (d) P (10 µM) with Al3+ (50 µM) and EDTA (100 µM) and then addition of 200 µM Al3+
.
18 #11 RT: 0.17 AV: 1 NL: 4.37E4
F: ITMS + c ESI Full ms [50.00-2000.00]
313.22
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
314.23
20
15
10
5
291.30
329.03
330.19
274.38
276.32
302.43
340.35
340
292.40
264.20
306.35
315.32
290.40
285.37
320.67
320
347.22
350
272.11
0
270
280
290
300
310
m/z
330
Figure S4. ESI-MS mass spectrum of P.
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Figure S5. 1 H NMR spectrum of P.
Figure S6. 13 C NMR spectrum of P.
3