A highly selective and sensitive turn-on probe for aluminum(III) based on quinoline Schiff's base and its cell imaging

Article abstract of DOI:10.1016/j.saa.2017.09.007

A reversible Schiff's base fluorescence probe for Al³⁺, (3,5-dichloro-2- hydroxybenzylidene) quinoline-2-carbohydrazide (QC), based on quinoline derivative has been designed, synthesized and evaluated. The QC exhibited a high sensitivity and se

Full text of DOI:10.1016/j.saa.2017.09.007

Accepted Manuscript
A highly selective and sensitive turn-on probe for aluminum(III)
based on quinoline Schiff's base and its cell imaging
Fenfen Zhou, Hongqing Wang, Pengying Liu, Qinghua Hu,
Yuyuan Wang, Can Liu, Jiangke Hu
PII:
DOI:
Reference:
S1386-1425(17)30721-7
doi: 10.1016/j.saa.2017.09.007
SAA 15441
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date:
Revised date:
Accepted date:
18 February 2017
2 September 2017
6 September 2017
Please cite this article as: Fenfen Zhou, Hongqing Wang, Pengying Liu, Qinghua Hu,
Yuyuan Wang, Can Liu, Jiangke Hu , A highly selective and sensitive turn-on probe for
aluminum(III) based on quinoline Schiff's base and its cell imaging, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.09.007
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ACCEPTED MANUSCRIPT
A highly selective and sensitive turn-on probe for
aluminum(III) based on quinoline schiff’s base and its cell
imaging
Fenfen Zhou, Hongqing Wang,* Pengying Liu, Qinghua Hu, Yuyuan Wang, Can Liu and Jiangke
Hu
College of Chemistry and Chemical Engineering, University of South China, 28 Changsheng
West Road Hengyang, Hunan 421001, PR China
*Corresponding Author: Hongqing Wang
E-mail: hqwang2015@126.com
Abstract
A reversible schiff's base fluorescence probe for Al3+, (3,5-dichloro-2-
hydroxybenzylidene) quinoline-2-carbohydrazide (QC), based on quinoline derivative
has been designed, synthesized and evaluated. The QC exhibited a high sensitivity
and selectivity toward Al3+ in EtOH-H2O (v/v=1:9, pH=6) by forming a 1:1 complex
with Al3+ and the detection limit of QC for Al3+ was as low as 0.012 µM.
Furthermore, these results displayed that the binding of QC-Al3+ was broken by F-, so
this system could be used to monitor F- in the future. The enhancement fluorescence
of the QC could be attributed to the inhibition of PET and ESIPT and the emergency
of CHEF process induced by Al3+. More importantly, QC was not only successfully
used for the determination of trace Al3+ in the tap water and the human blood serum,
but was valid for fluorescence imaging of Al3+ in the Hela cells.
Keywords：fluorescence probe，aluminum ions，schiff's base，cell imaging
1. Introduction
There are huge metal elements contained in earth and aluminum is the most
affluent one. Aluminum compounds play a significant role in daily life[1], industry[2]

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and agriculture[3]. Excessive Al3+ ions are not only harmful to growing plants[4], but
may give rise to a lot of illnesses, for example, Parkinson's disease[5], Alzheimer's
disease[6] and osteoporosis[7]. On the other hand, after absorption, aluminum would
accumulate in the body[8]. In this way, although trace amounts of aluminum ions
exist in the drinking water, low-dose chronic exposure to the ions may cause damage.
So, the detection of Al3+ has become research focus recently [9-11]. Among various
methods, the fluorescent probes has attracted more significant interests[8-10, 12, 13]
，due to its several outstanding advantages of simplicity, rapidity, high sensitivity
and selectivity[14, 15]. But, it is more difficult to recognize Al3+ than other metal ions
by fluorescence method because of its poor coordination ability and spectroscopic
characteristics[16]. Generally, Al3+ ions, as a hard acid, tend to coordinate with
ligands with hard-base donor sites. Moreover, the Schiff’s base with rich
nitrogen-oxygen coordination can offer a hard-base environment, which will provide
enough binding sites and improve the identification of probe for hard-acid metal [17].
Recently, a lot of Schiff’s base compounds have been reported as a probe for the
detection of aluminum ion by fluorescence [13, 18-23]. The majority of the reported
Al3+ probes, however, have disadvantages including only application in water
sample[24] or cell imaging [8, 19] and/or existing interference from other metal ions
(such as, Fe3+, Ni2+, Cu2+)[19, 20] and/or the higher rate of organic solvent in the
detection system[13, 21-23] and/or higher detection limit[13, 19]. Along those lines,
those probes were restricted application of water samples and biological systems.
Meanwhile, a fluorescence probe based on two or more mechanisms may be more
valuable because it can provide multiple signals and improve selectivity and
sensitivity[25-27]. But most of the recent years reported probes for Al3+ are
established on single signal mechanism[16, 28-30], which is intramolecular charge
transfer (ICT)[30], photo induced electron transfer (PET)[28] and excited-state
intramolecular proton transfer (ESIPT)[29]. For now, the development of the
fluorescence probe for Al3+ in water solution and cell with high selectivity and
sensitivity remains a difficult challenge. Obviously, Schiff's base ligands with a

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proton acceptor (–C=O, –N=) and a proton donor (–OH, –NH–) group can produce
the ESIPT process[31]. In the meantime, lone pair electrons from the nitrogen atom
(–C=N) of Schiff's base ligands can induce PET process[32]. In addition, fluorescence
probe with Schiff's base chelated metal ions would exhibit a chelation-enhanced
fluorescence (CHEF) process[33]. And the quinoline is usually used as a plotform to
construct probe for metal ion. Modifying the quinoline could easily give the probe
with the group of –OH, –NH–,–C=O, –N=, –C=N[34], which could not only satisfy a
prerequisite for the design of a probe based on more mechanisms but also improve the
water solubility of fluorescent probe.
Keeping these in mind, a novel schiff's base fluorescence probe
(3,5-dichloro-2-hydroxybenzyli-dene) quinoline-2-carbohydrazide(QC) has been
designed for Al3+. The fluorescent probe QC showed a high selective toward Al3+.
Furthermore, the binding of QC-Al3+ could be reversed by F-. More importantly,
these results revealed that the probe QC was used to detect Al3+ in human blood
serum and image Al3+ in HeLa cells.
2. Experimental
2.1 Equipment and material
The spectra of UV-Vis and fluorescence were gained through a Hitachi U-3900
(Japan) and a Hitachi F-7000(Japan), respectively, with a 1cm length and 4mL quart
optical cell. 1H NMR was taken on a Bruker 400MHZ in CDCl3 or DMSO-d6 with
TMS as the internal standard (Switzerland). The spectra of mass were recorded on
Thermo Fisher scientific LTQ FT Ultra mass spectrometer (America). The melting
point was determined on X-4 microscope melting point (China). The analyses of
elementary were realized by a Vario EL Ⅲelementary analysis instrument (Germany).
The pH was obtained on a Model pHs-3C meter (China). The imaging of HeLa cells
was gained on a Nikon ECLIPSE TE2000-S inverted fluorescence microscope
(Japan). The toxicity of HeLa cells was confirmed with Tecan Infinite F50 Microplate

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Reader (Germany). All chemicals of analytical grade reagent purchased form Aladdin.
Metal ions and anions solution were prepared from the corresponding nitrate salts and
sodium salts, respectively. The certified reference materials (CRMS) GBW(E)
081531 (liquid) and (CRMS) GBW(E) 080219 (liquid) were purchased from the
National Research Center for Certified Reference Materials (Beijing, China). Tap
water sample was got from our lab tap directly. All CRMS and water samples were
analyzed without pretreatment. The serum was pretreated by the method that has been
reported in the literature [35]. HeLa cells grew on a Cell Culture Flask with
Dulbecco’s Modified Eagle’s Medium, high glucose (H-DMEM) contained 10 %
Fetal Bovine Serum (FBS) at 37 ˚C and 5 % CO2 atmosphere. Double-distilled water
was employed in all experiments.
Stock solution (1.00×10-4 mol/L) of QC was prepared in ethanol as mother
samples. Stock solutions (1.00×10-3 mol/L) of Cu2+, Mg2+, Ba2+, Cr3+, Zn2+, K+, Bi3+,
Ag+, Al3+, Cd2+, Pb2+, Ca2+, Hg2+, Co2+, Ni2+, Fe2+, Mn2+, Fe3+, Na+ and related anions
were prepared in double distilled water. The following procedure was employed for
the all measurements. To 10 mL volumetric flasks containing different amounts of
metal ions, the proper amounts of the solution of QC was added directly, then diluted
ethanol-aqueous with Hexamethylenetetramine-HCl buffer solutions to 10 mL. After
shaking for 10 s and waiting for about 2 min at room temperature, 2-3 mL of the
solution was put into the fluorescent or UV-Vis cell and the absorption and
fluorescence were run. Fluorescence measurements were carried out with excitation
and emission slit width of 5 nm and excitation wavelength was 393 nm. Each value
was mean of three replicates.
2.2 Synthesis
2.2.1 Synthesis of quinoline-2-carbonyl chloride (compound 1)
Dichlorosulfoxide (12 mmol, 1.428 g) and pyridine (2.0 mL), in turn, were added
drop-wise to quinoline-2-carboxylic acid (12 mmol, 2.067 g) in 30 mL N,N-

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dimethyformamide (DMF) at 0 ℃and under N2 atmosphere. Then the solution
refluxed for 11 h. After removing the solvent under reduced pressure, the crude solid
was dried and recrystallized from petroleum to give 1.885 g of white solid of
quinoline-2-carbonyl chloride (compound 1) (Scheme 1.). (yield, 82 %; m.p. 97-98
℃). 1H NMR (400 MHz, DMSO˗d6) δ (ppm) 8.55 (d, J = 8.4 Hz, 1H, Ar–H), 8.17 (m,
1H, Ar–H), 8.07-8.02 (2H, Ar–H), 7.97-7.74(m, 1H, Ar–H), 7.72 (t, J = 7.5 Hz, 1H,
Ar–H).
2.2.2 Synthesis of quinoline-2-carboxylic hydrazide (compound 2)
Hydrazine hydrate (10 mmol, 0.412 g) was added to a methanolic solution (50 mL)
of quinoline-2-carbonyl chloride (6 mmol, 1.149 g) by droplet. Then the mixture
refluxed for 4.5 h. After removing the solvent under reduced pressure, the crude solid
was dried and recrystallized from methanol, 0.711 g yellow solid (quinoline-2-
carboxylic hydrazide, compound 2) was obtained (Scheme 1.). (yield,63%, m.p.
227–228 ℃).1H NMR (400 MHz, CDCl3) δ(ppm) 9.22 (s, 1H, –NH–), 8.35 (d, J =
8.5 Hz, 1H, Ar–H), 8.28 (d, J = 8.5 Hz, 1H, Ar–H), 8.11 (d, J = 8.5 Hz, 1H, Ar–H),
7.92 (t, J = 10.5 Hz, 1H, Ar–H), 7.65 (t, J = 7.2 Hz, 1H, Ar–H), 7.28 (s, 1H, Ar–H),
4.16 (s, 2H,–NH2).
2.2.3 Synthesis of QC
A methanolic solution (40 mL) of quinoline-4-carboxylic hydrazide (4 mmol, 0.748
g) was dropped tomethanolic solution (20 mL) of 3,5-dichlorosalicylaldehyde (0.969
g, 5 mmol) and kept the reaction mixture refluxing for 5 h under N2 atmosphere. After
removing the solvent under reduced pressure, the mixture was put into refrigerator for
overnight. Then 1.120 g of pale yellow crude solid was obtained by filtration. And the
pure QC was gotten by silica gel column chromatography (Scheme 1.). (yield, 78%,
m.p. 279–280 ℃) IR (KBr disk cm-1): 3309(–NH–), 3078(–OH), 1693, 1448, 1278,
1182(–C–O–C–), 1157, 1143, 771, 732(–C–Cl). 1H NMR (400 MHz, DMSO-d6)
δ(ppm) 12.89 (s, 1H,–NH–), 12.52 (s, 1H,Ar–OH), 8.86 (s, 1H, CH–), 8.61 (d, J = 8.5
Hz, 1H, Ar–H), 8.20 (d, J = 8.5 Hz, 2H, Ar–H), 8.10 (d, J = 7.4 Hz, 1H Ar–H), 7.90

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(t, J = 7.2 Hz, 1H, Ar–H), 7.80-7.63 (m, 1H, Ar–H), 7.62 (d, J = 2.4 Hz, 1H, Ar–H),
7.59 (d, J = 2.5 Hz, 1H, Ar–H). Elemental analiysis: C17H11Cl2N2O2; Calc., C, 56.69;
H, 3.08; N, 11.67; Found, C, 56.72; H, 3.10; N, 11.67. HRMS (MALDI): m/z
(M+1)+,359.82.
N2H4
SOCl2
H
N
refluxed
refluxed
COOH
N
NH2
N
COCl
N
O
compound 1
compound 2
Cl
Cl
compound 2
OH
Cl
H
N
N
N
HO
O
CHO
QC
Cl
Scheme 1. The route of synthesis for QC.
3. Results and discussion
3.1 Absorption spectra
The absorption spectra of QC (10 µM) with Al3+ (2.0 equiv.) or other metal ions
(2.0 equiv.) were measured in EtOH-H2O (v/v=1:9, pH=6.0). In the absence of Al3+,
the solutions of QC produced absorption bands at 313 nm and 340 nm, which could
be attributed to a π-π* transition of quinoline, favoured by the planar orientation
enforced by the intramolecular hydrogen bonding[36]. Because the intramolecular
hydrogen bond was broken after Al3+ added, absorption bands at 313 nm and 340 nm
red shifted to 330 nm and 393 nm, severally. Furthermore, other ions (including Cu2+,
Mg2+, Ba2+, Cr3+, Zn2+, K+, Bi3+, Ag+, Cd2+, Pb2+, Ca2+, Hg2+, Co2+, Ni2+, Fe2+, Mn2+,
Fe3+ and Na+) led to no absorption at 393 nm. But the absorption of QC with Al3+ at
330 nm was influenced by other metal ions (Fig. 1a.). Two legible isosbestic points
were obtained at 349 nm and 365 nm, respectively, with increasing the concentration
of Al3+, which explicitly implied the formation of QC-Al3+ and indicated a balanced
between QC with QC-Al3+ in solution[37]. Moreover, the absorbance at 393 nm

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enhanced gradually with the increasing concentration of Al3+ until 1.6 equiv. (Fig.
1b.).
Fig. 1(a). Absorption spectra of QC (10 µM，v/v=1:9, pH=6）with different metal ions (20 µM),metal ions
including Cu2+, Mg2+, Ba2+, Cr3+, Zn2+, K+, Bi3+, Ag+, Cd2+, Pb2+, Ca2+, Hg2+, Co2+, Ni2+, Fe2+, Mn2+, Fe3+ and Na+.
(b) Absorbance spectral changes of QC (10 µM) up addition of Al3+ (0.0-2.0 equiv.).
3.2 Fluorescence spectra
The fluorescence absorption of QC (10 µM) with and without Al3+ were
investigated in EtOH-H2O (v/v=1:9, λex=393 nm) during different pH conditions (Fig.
2.). Without Al3+, the weak fluorescence intensity of QC solution could be observed
from pH 3.0 to 9.0. Upon addition of Al3+ (2.0 equiv.), the fluorescence intensity was
dramatically enhanced at pH range from 3.0 to 8.0. Among of them, the fluorescence
intensity gradually increased from pH 3.0 to 4.5, attained a plateau from pH 4.5 to 7.0
and then decreased dramatically when the pH values are higher than 8.0. It ascribed
that QC binding Al3+ would be prevented at low pH value and at high pH values,
which resulted in weak fluorescence. In the absent of Al3+, there are PET and ESIPT
process for free QC, which induced by the transfer of the lone pair electrons from
nitrogen of –C=N and the formation of a hydrogen-bonding configuration[38] , so the
QC showed weak fluorescence. But at present of Al3+, Al3+ would form a complex
with QC, which formed effective restraint of the ESIPT and PET process and
increased the CHEF process [39] and produced enhancement fluorescence . In current
work, pH 6.0 with Hexamethylenetetramine-HCl butter solution was applied to the
whole experiment except for cells imaging.

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Fig. 2. Variation of fluorescence intensity of QC (10 µM) in EtOH-H2O (v/v=1:9,
pH=6, λex=393 nm) with and without Al3+ (2.0 equiv. ) as a function of pH.
Under the same conditions, the fluorescence response of QC was performed for 2.0
equiv. Al3+ or 2.0 equiv. other metal ions (including Cu2+, Mg2+, Ba2+, Cr3+, Zn2+, K+,
Bi3+, Ag+, Cd2+, Pb2+, Ca2+, Hg2+, Co2+, Ni2+, Fe2+, Mn2+, Fe3+ and Na+) (Fig. 3.). It
turned out that the fluorescence intensity was obviously enhanced only in the present
of Al3+ at 493 nm. However, no significant fluorescence changed was observed when
other metal ions were added. Furthermore, competition experiments (Fig. 4.), which
the solution of Al3+ (2.0 equiv.) and other individual cation (10.0 equiv.) were added
to probe QC (10 µM), were also manifested that the fluorescence intensity of
QC-Al3+ is rarely affected by the other tested metal ions. It ulteriorly proved that the
QC possess high sensitivity and selectivity to Al3+.

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Fig. 3. Fluorescence spectra of QC (10 µM) with Al3+ (2.0 equiv.)
and various metal ions (2.0 equiv.) in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm).
Fig. 4. Selective responses of QC (10 µM) for Al3+(2.0 equiv.) in the presence of various competitive metal ions
(10.0 equiv.) in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm). Black bars represent the addition of 10.0 equiv. of other
metal ions to a 10µM solution of QC or free QC. Red bars represent fluorescence intensity of a mixture of QC (10
µM) with other metal ions (10.0 equiv.) in followed by addition of 2.0 equiv of Al3+ to the solution，
respectively.
.
To further study the recognizing abilities of QC toward Al3+, fluorescence titrations
were carried out. As shown in Fig. 5., the free QC (10 µM) emitted weak
fluorescence at 493nm in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm). With the
gradually increasing concentration of Al3+, the fluorescence intensity at 493 nm
continuously enhanced until concentration of Al3+ reached 1.6 equiv. As well as, the
fluorescence intensity of QC-Al3+ solution was linear dependence to the concentration
of Al3+ in the range from 0 µM to 10 µM with the correlation coefficient of
R2=0.9989 (Fig. 5. inset). On the basis of these relationships and 3σ/K, where K was
the slope between fluorescence intensity and sample concentration of Al3+ at 493 nm,
σ was the standard deviation of blank solution, the detection limit was estimated about
0.012 µM (Table 1.). Meanwhile, probe QC has water solubility, so it could be
applied for water sample and cell imaging. These consequences exhibited that the
probe QC will play a crucial role in the field of environment monitoring.

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Fig. 5. Fluorescence spectra of QC (10 µM) with addition of increasing amount of Al3+ (0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 14, 16, 18, and 20 µM) in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm). Inset: fluorescence intensity at
493 nm versus the number of concentration of Al3+ added.
Table 1. The comparison of QC with some other probes for Al3+ ions.
Solution
mechanism
LOD(µM)
Application
Reference
(v/v)
FRET
PET
aqueous
0.50
0.19
/
[12]
[13]
[21]
[22]
[23]
[39]
[34]
ethanol
/
CHEF
PET
DMSO-H2O 4:6
ethanol
0.027
0.10
/
/
PET
ethanol
0.027
0.10
/
FRET
PET,CHEF
EtOH-H2O 1:99
ethanol
Cell imaging
/
0.082
CH3OH-H2O
95:5
CHEF
0.60
2.40
/
[40]
[41]
ESIPT
ESIPT,PET,
CHEF
DMSO
Cell imaging
Water samples
and Cell imaging
EtOH-H2O 1:9
0.012
Present work

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3.3 Binding mode of QC and Al3+
To investigate the binding mechanism between QC and Al3+, the Job’s plot for the
fluorescence intensity was tested by sustaining the total concentration of 10 μM for
QC mixed Al3+ and altering the mole fraction of Al3+ from 0 to 0.9 in EtOH-H2O
(v/v=1:9, pH=6.0) (Fig. 6.). When the molar fraction reached 0.5, the highest
fluorescence intensity was obtained, which might informed that the stoichiometric
binding mode for QC-Al3+ was 1:1 in the media [42, 43]
.
Fig. 6. Job’s plot of QC with Al3+ in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm). Total concentration of QC
and Al3+ was maintained constant at 10µM and the mole fraction of Al3+ changed.
Furthermore, the recognition reversibility is a very significant research. The
reversibility of complex QC-Al3+ were investigated through various anion (3.0equiv.),
2-
3-
2-
-
-
such as I-, F-, Br-, Cl-, Ac-, CO3 , PO4 , S2-, SO4 , ClO4 and NO3 in EtOH-H2O
(v/v=1:9, pH=6.0). As shown in Fig. 7., the results showed that only F- produced
obvious fluorescence quenching at 493 nm, which was ascribed to the F- reversed the
process of the chelation of QC-Al3+. The accomplish time of the reversible process
was 2 minutes. Fortunately, this cycle process could be repeated multiple times
without loss of fluorescence intensity.

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Fig. 7. Fluoresence spectra of QC-Al3+with different anions (3.0 equiv.).
-
-
Anions include I-, F-, Br-, Cl-, Ac-, CO32-, PO43-, S2-, SO42-, ClO4 and NO3 .
Benesi-Hildebrand method was also used to confirm the binding stoichiometry.
When hypothesizing a 1:1 associate-ion between QC and Al3+, the binding constant
was calculated from the fluorescence intensity data according to the
Benesi˗Hildebrand equations[44, 45]:
Fmax − Fmin
F − Fmin
1
=
+ 1
3+
[
]
Ka Al
Fmin was the fluorescence intensity of the free QC. F was the fluorescence intensity
of QC-Al3+ at any intermediate aluminum concentration. Fmax was the fluorescence
intensities of the complete interaction of QC binding aluminum. Ka was the binding
constant (M-1) for Al3+, [Al3+] represent the concentration of Al3+. The intercept of 1
and the slope of 1/Ka would be expected in a regression plot of (Fmax - Fmin)/(F - Fmin)
vs. 1/[Al3+]. As the plot of titration profiles showed, excellent linearity was exhibited
1:1 binding mode between QC and Al3+ (Fig. 8.), and Ka was calculated as 8.44×104
M-1 based on slope and intercept.

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Fig. 8. Benesi-Hildebrand plot of QC (10 µM) at different concentration Al3+ (0-10 µM)
in EtOH-H2O (v/v=1:9, pH=6, λex=393 nm).
To further define the bonding formation of QC-Al3+, the 1H NMR titration
experiments were executed with or without Al3+ in DMSO-d6. In 1H-NMR titration,
Al3+ was put into the DMSO-d6 solution of QC and spectral change was obtained
(Fig. S1, Supporting information). These peaks at 12.89 ppm and 12.52 ppm should
respectively represent the labile protons of –NH– and –OH. The proton peak of the
–OH at 12.52 ppm vanished and the proton signal of the imine (–NH–) at 12.89 ppm
slightly receded while Al3+ existed. When 3.0 equiv. of F- was added, the peak of
–OH at 12.52 ppm and –NH– at 12.89 ppm were recovered. These results might
informed that QC may chelate Al3+ by interaction with oxygen atoms from –C=O and
–OH of the salicylaldehyde hydroxyl group and nitrogen atom of –C=N [17, 46]
(Scheme 2.). Namely, the change of QC fluorescence intensity in the case of Al3+ ion
was owed to the formation of QC-Al3+ complex through two oxygen of carboxylate
and phenol groups respectively, and nitrogen of imine moiety with Al3+, which
formed effective restraint of the ESIPT and PET process and increased the CHEF
process[33].

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Scheme 2. Proposed mechanism for detection of Al3+ and F- by QC
3.5 The preliminary application of QC
To verify the accuracy of the fluorescence spectra method for QC detect Al3+, two
certified reference materials (CRMs)(GBW(E)081531 GBW(E)080219) were used.
These analytical results matched the standard values of CRMs (Table 2.). It was easy
to see from Table 2., these values (error rates 5%) of Tmeasurement were smaller than the
corresponding Treferences values[47], which indicated fluorescence spectra method is
reliable. Furthermore, to evaluate unknown concentration of Al3+ in the real samples,
standard addition method was used with Al(NO3)3 (GBW(E) 080219) as standard
material (Table 3.). The results showed that the concentrations of Al3+ were found to
be 0.25 ± 0.033 µM and 2.10 ± 0.11 µM in the volunteer serum and dialysis patient
serum, respectively, which were consistent with the reference[48] , and 2.86 ± 0.033
µM in the tap water. At the same time, the recovery rates of Al3+ were in the range of
98.6%–102.3%. So, the provided detection method for Al3+ possesses high accuracy
and precision.
Table 2. Analytical results for the detection of Al3+ in certified reference materials.
Concentration of Al3+(µg/mL)
T-test
Sample
Certified
100
Measured
99.983
T(references)[47]
4.303
T(measurement)
GBW(E) 081531
GBW(E) 080219
0.536
100
99.9962
4.303
1.068

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Table 3. Analytical results for the detection of Al3+ in serum and tap water samples.
Concentration of Al3+(µM/L)
Sample
Recovery Rate(%)
Added of Al(NO)3
Measured
0.25±0. 021
2.22±0.045
4.24±0.034
6.25±0.010
2.20±0.12
4.19±0.15
6.21±0.09
8.21±0.10
2.86±0.015
4.85±0.035
6.91±0.027
8.90±0.056
Volunteer serum
0.00
2.00
4.00
6.00
0.00
2.00
4.00
6.00
0.00
2.00
4.00
6.00
0.0
99.5
101.2
98.6
0.0
dialysis patient serum
99.6
102.3
98.9
0.0
tap water
100.7
101.3
98.9
3.6 Cell studies of QC in the presence of Al3+
To test the cytotoxicity of probe QC and Al3+, these HeLa cells were incubated
with different concentration of probe QC (0 to 60 µM) and Al3+ (1 µM – 100 µM).
Then cell viability was determined by an MTT assay. As shown in Fig. 9., these
results represented no toxicity to HeLa cells in the present of 0-10 µM QC and 1-10
µM Al3+.

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Fig. 9. The cytotoxity of QC in HeLa cells.
Subsequent experiments were aimed to evaluate whether QC could be used to
fluorescently visualize Al3+ in living matrices. Hela cell were first incubated with 10
μM solution of probe QC for 30 min at 37 °C and washed twice with PBS. Finally,
Hela cells (or the treated cells) exposed to 10 μM solution of Al3+ for another 30 min
at 37 °C and washed with H-DMEM before imaging. As shown in the Fig. 10., no
intracellular fluorescence (Fig. 10a., 10b.) was observed when the cells were only
treated with solution of QC. While strong fluorescence (Fig. 10e.) was observed in
cells exposed to QC and Al3+. Cells that were exposed to Al3+ did not displayed
fluorescence (data not shown). And the bright-field transmission image of cells treated
with QC or Al3+ (Fig. 10a. and d.) confirmed that the cells were viable throughout the
imaging experiments. These results indicated that probe QC was cell permeable and
may be used to detect Al3+ in biological sample.

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Fig. 10. Bright field (a) and fluorescence image (b) of HeLa cells (40X) incubated with probe QC (10 µM)
for 30 min; Bright field (d) and fluorescence image (e) of HeLa cells (40X) incubated with probe QC (10 µM) and
Al3+ (10 μM) for 30 min. Fluorescence image taken from (b & e) green channel (480 nm -530 nm). (c) overlay
image of (a) - (b); (f) overlay image of (d) - (e).
4. Conclusions
In a word, a novel turn on fluorescence probe QC based on quinoline has been
developed, which showed high selectivity and sensitivity in the present of
concomitant metal ions (including: Cu2+, Mg2+, Ba2+, Cr3+, Zn2+, K+, Bi3+, Ag+, Cd2+,
Pb2+, Ca2+, Hg2+, Co2+, Ni2+, Fe2+, Mn2+, Fe3+ and Na+) via a 1:1 binding mode with
the binding constant being 8.44 × 104 M-1 in EtOH-H2O (v/v=1:9, pH=6). And F- can
reverse the fluorescence of QC-Al3+, which would be used to test F- in the same
system. In addition, the interaction mechanism of QC and Al3+ was proposed. An
effective fluorescence analysis method for Al3+ has been set up with the concentration
of Al3+ in the range of 0-10 µM, and the detection limit was found to be 0.012 µM.
Finally, the probe QC indicated the worth for test of intracellular Al3+. Therefore, the
probe QC displays strong potential for applications in environmental monitoring and
biological diagnostics analysis.

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Acknowledgments
This work was financially supported by National Natural Science Foundation of China
(No. 11175080), Natural Science Foundation of Hunan Province (No. 2016JJ5003), Science
Foundation of Hunan Province Department of Education (No. 12A117), Hunan Provincial
Innovation Foundation for Postgraduate (No.CX2016B440)
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Graphical abstract

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Highlights
1) A novel turn on fluorescent probe for aluminum(III) based on asymmetric
quinoline Schiff's base has been synthesized.
2) The fluorescent probe QC, which could apply in water sample, serum and cell
image, has higher selectivity and lower limit of detection for Al3+ comparing with the
previous reports.
3) Binding mode and coordination sites of probe QC with Al3+ were proposed.
4) The rate of water in the solvent was increased.