Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand

Article abstract of DOI:10.1016/j.saa.2017.02.048

A novel adenine (Ad) fluorescence probe (Eu^III-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the Eu^III-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV–vis) and fluorescence spectroscopy. When the Eu^III-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the Eu^III-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using Eu^III-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of Eu^III-dtpa-bis(guanine) at 570?nm as a function of adenine (Ad) concentration in the range of 0.00–5.00?×?10^??5?mol?L^??1 was observed. The detection limit is about 4.70?×?10^??7?mol?L^??1.

Full text of DOI:10.1016/j.saa.2017.02.048

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Design and synthesis of novel adenine fluorescence probe based on
Eu(III) complexes with dtpa-bis(guanine) ligand
b,
Fengyun Tian a, Xiaoqing Jiang a, Xuekai Dou b, Qiong Wu a, Jun Wang a, , Youtao Song
⁎
⁎
a
College of Chemistry, Liaoning University, Shenyang 110036, PR China
b
College of Environment, Liaoning University, Shenyang 110036, PR China
a r t i c l e i n f o
a b s t r a c t
A novel adenine (Ad) fluorescence probe (EuIII-dtpa-bis(guanine)) was designed and synthesized by improving
experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand
was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III)
complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel
fluorescence probe, the EuIII-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong
targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this
probe in buffer solution was studied by ultraviolet-visible (UV–vis) and fluorescence spectroscopy. When the
EuIII-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was sig-
nificantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and
uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the EuIII-dtpa-bis(guanine) solu-
tion, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu),
xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using EuIII-dtpa-
bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of
adenine (Ad). A linear response of fluorescence emission intensities of EuIII-dtpa-bis(guanine) at 570 nm as a
function of adenine (Ad) concentration in the range of 0.00–5.00 × 10−5 mol L−1 was observed. The detection
Article history:
Received 29 June 2016
Received in revised form 17 February 2017
Accepted 21 February 2017
Available online 24 February 2017
Keywords:
Design and synthesis
Adenine fluorescence probe
Aminopolycarboxylic acid ligand
Eu(III) complex
Fluorescence spectroscopy
limit is about 4.70 × 10−7 mol L−1
.
© 2017 Published by Elsevier B.V.
1. Introduction
In recent years, a variety of methods including high-performance
liquid chromatography, ion-pairing liquid chromatography, micellar
electrokinetic chromatography, electrochemistry, capillary electropho-
resis, chemiluminescence, spectrophotometry, surface enhanced
Raman scattering and mass spectrometry have been developed for the
determination and quantification of adenine (Ad) in biological fluids
[12–26]. In fact, these analytic methods were thought to be rapid and
sensitive, but they also have several shortcomings such as high cost,
high time consumption, tedious pretreatment steps and poor capture
ability to substrates. Therefore, it is necessary to develop new method
to detect adenine (Ad). Compared to these methods, the fluorescence
probe methods not only have characteristics of high sensitivity, high ac-
curacy and low detection concentration, but also have features of high
specificity and strong targeting property [27–30]. It is well known that
some Eu(III) complexes are successfully applied in materials science
field as luminescent probe with their high and sharply spiked fluores-
cence emission efficiency, long lifetime and large Stokes shift [31–33].
Aminopolycarboxylic acid such as dtpa as a good chelating agent can
form extraordinarily stable complexes with many rare earth metal
ions and emit a specific fluorescence under excitation of light of appro-
priate wavelength [34]. However, as a good fluorescent probe, in addi-
tion to possessing obvious photo-responsive property, it should have
Adenine (Ad) is an important component of nucleic acid which is not
only participate in the synthesis of genetic material playing a crucial role
in life process and fulfilling a variety of functions in the metabolism of
the cell, but also has widespread influences on coronary and cerebral
circulation [1–3]. It can also control of blood flow, prevention of cardiac
arrhythmias, inhibition of neurotransmitter release and modulation of
adenylate cyclase activity [4]. In addition, adenine (Ad) can be used as
drugs for leukopenia caused by tumor chemotherapy disease, also
used in acute granulocytopenia. The abnormal change of adenine (Ad)
in organism may indicate the mutation and deficiency of the immune
system leading to the manifestation of various diseases including epi-
lepsy, cancer, tumorigenesis, mental retardation, HIV infection,
Parkinson's disease, carcinoma and liver diseases [5–10]. Therefore,
the determination of concentration level of adenine (Ad) is considered
to be a challenging and important task in the field of physiology and
clinical pathology [11].
⁎
Corresponding authors.
E-mail addresses: wangjun888tg@126.com (J. Wang), wangjun891@sina.com
(Y. Song).
http://dx.doi.org/10.1016/j.saa.2017.02.048
1386-1425/© 2017 Published by Elsevier B.V.

F. Tian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
195
good specificity, high recognition ability and strong choice ability. The
2.2. Material
Eu(III) complexes with some aminopolycarboxylic acid ligands can be
used as fundamental fluorescence probes due to the obvious photo-re-
sponsive property. In order to make these fundamental fluorescence
probes have better recognition ability and strong choice ability, it is nec-
essary to pointedly modify the dtpa ligand by using some targeting
compounds to detected object.
Diethylenetriamine pentaacetic acid (dtpa) and guanine (Gu) (A.R.,
Beijing SHLHT Science & Trade Co., Ltd., China) were purchased and
used to synthesize the dtpa-bis(guanine) ligand. Adenine (Ad), hypo-
xanthine (Hy), xanthine (Xa) and uric acid (Ur) (A.R., Beijing SHLHT Sci-
ence & Trade Co., Ltd., China) were purchased and used to be target
client. Eu(NO3)3·6H2O (99.999%, Yuelong Rare Earth Co., Ltd., China)
was obtained to prepare the EuIII-dtpa-bis(guanine) complex as fluores-
cent probe. Anhydrous acetic anhydride and DMF (analytical purity,
Shenyang Chemical Reagent Plant, China) were purchased and used as
solvent. Pyridine and Triethylamine (analytical purity, Shenyang Chem-
ical Reagent Plant, China) were obtained and used as acid-binding-
agent. Tris (hydroxyl-methyl) aminomethane (Tris) and HCl (analytical
purity, Shenyang Chemical Reagent Plant, China) were used to prepare
the Tris-HCl (pH = 7.4 and [Tris-HCl] = 50 mmol L−1) buffer solution
in order to maintain the ionic strength and adjust the solution acidity.
In this paper, the structure of dtpa was modified by guanine and
formed a new ligand dtpa-bis(guanine). The Eu3+ ion can form a
nine-coordinate EuIII-dtpa-bis(guanine) complex with the new ligand
dtpa-bis(guanine) [35–37]. In the EuIII-dtpa-bis(guanine) complex as
fluorescence people, two guanines at the two ends (up and down),
like the two arms. When the EuIII-dtpa-bis(guanine) encounters the ad-
enine, whose molecular structure and chemical composition are similar
to that of guanine, these two arms (guanines) can capture the adenine
tightly. Because of the change of ligands from water to adenine
forming the new coordination bond, the fluorescence intensity of
EuIII-dtpa-bis(guanine) was changed obviously. Due to the highly
chemical similarity, the EuIII-dtpa-bis(guanine) complex can com-
bine with adenine exclusively. Thus, as adenine fluorescence probe
the EuIII-dtpa-bis(guanine) complex not only have the characteris-
tics of high sensitivity, high accuracy and low detection concentra-
tion, but also the advantages of strong targeting, high recognition
ability and choice ability. Subsequently, by means of fluorescence
spectrum, it was found that the fluorescence emission intensity
was significantly enhanced, when the adenine was introduced to
the EuIII-dtpa-bis(guanine) solution. While the other base com-
pounds, such as guanine, xanthine, hypoxanthine and uric acid,
were added to the EuIII-dtpa-bis(guanine) solution, the fluorescence
emission intensity was hardly changed. Meanwhile, in this work, the
effects and interferences of guanine, hypoxanthine, xanthine and
uric acid were also studied. It was found that the EuIII-dtpa-
bis(guanine) complex as fluorescence probe can detect adenine
with specificity and not be affected by other base compounds. There-
fore, the sensitivity and specificity of the EuIII-dtpa-bis(guanine)
complex in the adenine detection could be confirmed.
2.3. Synthesis of Diethylenetriamine Pentaacetic Acid Dianhydride (dtpaa)
It must be pointed out that the diethylenetriamine pentaacetic acid
dianhydride (dtpaa) is demand for the start of all experiments and its
synthesis procedure is described in Scheme 1. Diethylenetriamine
pentaacetic acid (dtpa) (7.80 g, 0.02 mmol) was dissolved in acetic an-
hydride (8.00 mL, 0.08 mmol) and pyridine (10.00 mL, 0.12 mmol) as
acid-binding agent under anhydrous condition. The mixed solution
was stirred for one day under heat-refluxing at 65 °C. Afterwards, the
reaction mixture was cooled down to room temperature, and the sol-
vent was removed by reduced pressure filter. The residue washed
twice by acetic anhydride and anhydrous diethyl ether. Finally, the res-
idue was dried to give 6.50 g white powder under vacuum (52 kpa) at
80 °C with yield of 83%. FT-IR (KBr, cm−1): 1642.41, 1772.10, 1821.08,
2341.42, 2820.47 and 2979.80. 1H NMR (500 MHz, DMSO): d = 2.593
(t, 4H), 2.748 (t, 4H), 3.300 (s, 2H), 3.705 (s, 8H) and 11.013 (s, 1H).
2.4. Synthesis of dtpa-bis(guanine)
Dtpa-bis(guanine) ligand was synthesized by the aminolysis reac-
tion between dtpaa and guanine and the synthesis procedure is shown
in Scheme 1. Dtpaa (1.96 g, 55 mmol) was dissolved in DMF (50 mL)
and Trithylamine as base under anhydrous condition. Subsequently, the
guanine was added to the mixed solution slowly. The mixed solution
was stirred 24 h under heat-refluxing at 100 °C. The mixture was then
cooled down to room temperature. After the solvent was removed by vac-
uum filter, the white solid was obtained. The white solid was evaporated
to dryness under vacuum (52 kpa) at 50 °C to give 2.30 g white powdery
solid with yield of 92%. FT-IR (KBr, cm−1): 1668, 1710, 2691, 2854, 2908,
3119 and 3321. 1H NMR (500 MHz, DMSO): d = 2.010 (m, 2H), 2.717 (s,
2H), 2.807 (d, 2H), 2.460 (t, 8H), 3.254 (s, 4H), 3.301 (s, 6H), 7.503 (d, 2H),
7.973 (s, 2H) 8.010 (s, 2H) and 11.012 (s, 3H).
2. Experimental
2.1. Apparatus
Fourier Transform-Infrared (FT-IR) spectra were taken in KBr disks
on a Nicolet 5700 FTIR spectrometer. NMR spectra were conducted
with an Agilent Technologies Plus-400MR spectrometer with DMSO-
d6, D2O and NaOH-d1 as the solvent and tetramethysilane (TMS) as in-
ternal standard. Fluorescence determination experiments were carried
out by fluorophotometer (Cary 300, Varian Company, USA) and the
UV–vis absorption spectra were recorded with an UV–Vis spectropho-
tometer (Cary 50, Varian Company, USA).
Scheme. 1. The structure and synthetic route of the dtpa-bis(guanine) ligands.

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F. Tian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
2.5. Synthesis of the Eu(III) Complex With dtpa-bis(guanine) Ligand
0.0870 g (0.13 mmol) dtpa-bis(guanine) was added to 50 mL Tris-
HCl buffer solution. 0.0570 g (0.13 mmol) Eu(NO3)3·6H2O powder
was added to 50 mL Tris-HCl buffer solution. After that the two above
buffer solution were mixed together and refluxed and stirred for one
hour until solution became transparent, constanted volume in 250 mL
volumetric flask and kept in refrigerator 4.0 °C as stock solution to be
used.
2.6. Preparation of the Solution of Various Bases
0.017 g (0.13 mmol) adenine, 0.019 g (0.13 mmol) guanine, 0.019 g
(0.13 mmol) xanthine, 0.017 g (0.13 mmol) hypoxanthine and 0.021 g
(0.13 mmol) uric acid were dissolved in 20 mL Tris-HCl buffer solution
and constanted in 250 mL volumetric flask to obtain 5.00 × 10−4 mol L−1
solution, respectively. The stock solutions were kept in refrigerator
4.0 °C. The stock solutions were diluted to required concentrations
with Tris-HCl buffer solution when needed.
Fig. 2. UV–vis spectra of Eu(III) complexs (5.00 × 10−5 mol L−1) and Eu-dtpa-
bis(guanine) complex (5.00
×
10−5 mol L−1
)
and dtpa-bis(guanine)
(5.00 × 10−5 mol L−1) solution. (Eu-dtpa-DG: Eu-dtpa-bis(guanine)).
2.7. Determination of UV–Vis Spectra of Eu-dtpa-bis(guanine) solution
which displays a red-shift by 22 cm−1 compared with that 1397 cm−1
of dtpa. Furthermore, the vas(COO) of dtpa-bis(guanine) appears at
1710 cm−1, while the vas(COO) of dtpa appears at 1732 cm−1, so the
dtpa-bis(guanine) displays a red-shift by 22 cm−1. And that the
vas(CONH) of dtpa-bis(guanine) appears at 1668 cm−1 and the charac-
The dilute solution of EuIII-dtpa-bis(guanine) and dtpa-bis(guanine)
(5.00 × 10−5 mol L−1) were prepared in Tris-HCl buffer solution. The
solution of Eu3+ (5.00 × 10−5 mol L−1) was also prepared in Tris-HCl
buffer solution. These above solutions were put in a quartz cell
(10.0 mm width), and the absorption spectra were recorded at room
temperature.
teristic broad absorption peaks of hydroxy group appears at 3446 cm−1
.
These shifts confirmed that the dtpa-bis(guanine) was synthesized by
the acylation action between dtpaa and guanine.
2.8. Determination of Fluorescence Spectra of Eu-dtpa-bis(guanine) Solu-
tion With Adenine
3.2. UV–Vis Spectra of Eu-dtpa-bis(guanine) Solution
The UV–vis absorption spectra of dtpa-bis(guanine), EuIII-dtpa-
The dilute solution of EuIII-dtpa-bis(guanine), adenine, guanine, hy-
poxanthine, xanthine and uric acid (5.00 × 10−5 mol L−1) were pre-
pared in Tris-HCl solution, respectively. These above solutions were
placed in quartz cell (10.0 mm width) and all fluorescence spectra
were carried out at room temperature.
bis(guanine) and Eu(III) ion in Tris-HCl (pH = 7.4 and [Tris-HCl] =
[NaCl] = 50 mmol L−1) buffer solution solutions (5.00 × 10−5 mol L−1
)
were all shown in Fig. 2. It can be found that the Eu(III) solution gives
a maximum absorption peak at about 220 nm wavelength (λmax). And
that, for dtpa-bis(guanine) its solution displays three absorption peaks
at 214 nm, 242 nm and 274 nm, respectively. Upon the addition of
dtpa-bis(guanine), the formed EuIII-dtpa-bis(guanine) complex also
gives a maximum absorption peak at 220 nm, but slightly becomes
stronger compared with that of Eu3+ ion solution. The other absorption
peaks obviously become weaker compared with those of dtpa-
bis(guanine) solution. It indicates that the Eu3+ ion and dtpa-
bis(guanine) can change their electronic structure each other resulting
in the potential acting as fluorescence probe.
3. Results and Discussion
3.1. FT-IR Spectra of Eu-dtpa-bis(guanine)
Dtpaa was synthesized by the decarboxylic reaction of dtpa. And the
dtpa-bis(guanine) was synthesized by the acylation reaction between
dtpaa and guanine. All obtained compounds were characterized by 1H
NMR (the corresponding data were given in the experimental part)
and Fourier transform infrared spectra. The comparison of FT-IR spectra
among dtpa, guanine and dtpa-bis(guanine) is shown in Fig. 1. It can be
3.3. Fluorescence Spectra of Eu-dtpa-bis(guanine) Solution With Adenine
found that the v(C\\N) of dtpa-bis(guanine) appears at 940 cm−1
which displays a red-shift by 10 cm−1 compared with the 950 cm−1
of guanine. The vs(COO) of dtpa-bis(guanine) appears at 1418 cm−1
,
The fluorescence spectra of EuIII-dtpa-bis(guanine) complex and
corresponding mixed solutions with a series of base compounds, such
as adenine (Ad), guanine (Gu), hypoxanthine (Hy), xanthine (Xa) and
,
Fig. 1. Infrared spectra of guanine (Gu), diethylenetriamine pentaacetic acid (dtpa) and dtpa-bis(guanine). (Eu-dtpa-DG: Eu-dtpa-bis(guanine)).

F. Tian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
197
Fig. 3. Fluorescence spectra (λex = 280 nm ) (a) of Eu-dtpa-bis(guanine) in Tris-HCl buffer solution upon the addition of adenine (Ad), hypoxanthine (Hy), xanthine (Xa), uric acid (Ur)
and guanine (Gu) and the corresponding fluorescence intensities (b). ([Eu-dtpa-bis(guanine)] = [Ad] = [Hy] = [Xa] = [Ur] = [Gu] = 5.00 × 10−5 mol L−1, [Tris-HCl] = 50 mmol L−1
,
pH = 7.40 and Tsolu = 25.00 0.02 °C. Eu-dtpa-DG: Eu-dtpa-bis(guanine)).
uric acid (Ur), are depicted in Fig. 3. It can be found that, under the ex-
citation of ultraviolet light at 280 nm, the EuIII-dtpa-bis(guanine) com-
plex in aqueous solution emita moderately strong green fluorescence
at maximum wavelength at 560 nm owing to the presence of Eu3+
ion. When the adenine (Ad) was introduced to the EuIII-dtpa-
bis(guanine) solution, the fluorescence emission intensity was signifi-
cantly enhanced about 2.5 times. While with the addition of guanine
(Gu), hypoxanthine (Hy), xanthine (Xa) and uric acid (Ur) to the EuIII-
dtpa-bis(guanine) solution, the fluorescence emission intensity was ob-
viously decreased. That is, the guanine (Gu), hypoxanthine (Hy), xan-
bis(guanine) complex can be used as a selective fluorescence probe to
detect adenine (Ad).
3.4. Effects of Adding Other Bases on Fluorescence Spectrum of EuIII-dtpa-
bis(guanine) and Adenine (Ad) Solution
The effects of other bases including guanine (Gu), hypoxanthine
(Hy), xanthine (Xa) and uric acid (Ur) on the fluorescence of EuIII
-
dtpa-bis(guanine) and adenine (Ad) solution were researched. As
shown in Fig. 4, when adding guanine (Gu), hypoxanthine (Hy), xan-
thine (Xa) and uric acid (Ur) into the EuIII-dtpa-bis(guanine) and ade-
nine (Ad) solution, respectively, the fluorescence emission intensity
and peak position are hardly changed. It can be predicted that the pres-
ence of these base compounds will not disturb the detection and analy-
sis of adenine (Ad) due to their different interaction mode with EuIII-
dtpa-bis(guanine) complex. It indicates that the EuIII-dtpa-bis(guanine)
complex as fluorescence probe to detect adenine (Ad) exhibits high sen-
sitivity and specificity as well as good recognition ability. Moreover, it
can be confirmed that the use of guanine (Gu) combined with the
EuIII-dtpa forming EuIII-dtpa-bis(guanine) complex brings the specific
thine (Xa) and uric acid (Ur) can quench the fluorescence of EuIII
-
dtpa-bis(guanine) solution more or less and the quenched order is as
uric acid (Ur) ˃ xanthine (Xa) ˃ guanine (Gu) ˃ hypoxanthine (Hy). It
could be confirmed that a sensitive response of EuIII-dtpa-bis(guanine)
solution was observed when only adenine (Ad) was added, though
these base compounds have similar molecular structure and chemical
composition. In addition, it can be estimated that the adenine (Ad)
adopts a different interaction mode with EuIII-dtpa-bis(guanine) is
from other base compounds (guanine (Gu), hypoxanthine (Hy), xan-
thine (Xa) and uric acid (Ur). It suggested that the EuIII-dtpa-
Fig. 4. Fluorescence spectra (λex = 280 nm) (a) of Eu-dtpa-bis(guanine) and Eu-dtpa-bis(guanine) + A (adenine) in Tris-HCl buffer solution upon the addition of hypoxanthine (Hy),
xanthine (Xa), uric acid (Ur) and guanine (Gu) and the corresponding fluorescence intensities (b) at λem = 560 nm. ([Eu-dtpa-bis(guanine)] = [Ad] = [Hy] = [Xa] = [Ur] = [Gu] =
5.0 × 10−5 mol L−1, [Tris-HCl] = 50 mmol L−1, pH = 7.40 and Tsolu = 25.00 0.02 °C. Eu-dtpa-DG: Eu-dtpa-bis(guanine)).

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F. Tian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
Fig. 5. Fluorescence spectra (λex = 280 nm) (a) of Eu-dtpa-bis(guanine) in Tris-HCl buffer solution upon the addition of adenine (Ad) with different concentrations and linear responses
(b) of Eu-dtpa-bis(guanine) as a function of adenine (Ad) concentrations (0.00–5.00 × 10−5 mol L−1). ([Eu-dtpa-bis(guanine)] = 5.00 × 10−5 mol L−1, [Tris-HCl] = 50 mmol L−1, pH =
7.40 and Tsolu = 25.00 0.02 °C. Eu-dtpa-DG: Eu-dtpa-bis(guanine)).
targeting to adenine (Ad) due to their similar molecular structure and
chemical composition.
is well known that the Eu3+ ion can form nine-coordinate complexes
with various aminopolycarboxylic acid ligands [35–37]. For this newly
synthesized dtpa-bis(guanine) ligand, which is modified dtpa by two
guanines at its two ends (up and down), respectively, it also is an
eight-dentate ligand. Therefore, in the EuIII-dtpa-bis(guanine) complex,
as ninth coordination ligand one water molecule (H2O) should be coor-
dinated with EuIII ion. It was inferred that the Eu3+ is coordinated with
the three nitrogen atoms and five oxygen atoms from dtpa-
bis(guanine) ligand and one oxygen atom from H2O molecule in EuIII-
dtpa-bis(guanine) complex. Upon addition of the adenine (Ad), Because
the adenine (Ad) and guanine (Gu) both have flat molecular structure,
the π-π stacking binding will be formed between adenine (Ad) and gua-
nine (Gu) in EuIII-dtpa-bis(guanine) due to their similar molecular
structures and chemical compositions.
3.5. Fluorescence Spectra of EuIII-dtpa-bis(guanine) Solution With Adenine
(Ad) Concentration
It can be seen from Fig. 5(a) that the fluorescence emission intensity
is obviously enhanced after adding adenine (Ad), and then further in-
creases gradually with increasing adenine (Ad) concentration. It indi-
cates that this fluorescence change of EuIII-dtpa-bis(guanine) complex
may be used for the detection and analysis of adenine (Ad). In addition,
a linear response of fluorescence intensities of EuIII-dtpa-bis(guanine)
solution as a function of adenine (Ad) concentration at 570 nm was
also observed in Fig. 5(b) in the range of 0.00–5.00 × 10−5 mol L−1
.
The corresponding linear equation is y = 32.085x + 358.07 (R =
0.9758), where y is the fluorescence intensity at 570 nm measured at
a given adenine (Ad) concentration and x represents the concentration
of added adenine (Ad). According to the formula of LOD (limit of detec-
Figuratively, like two arms the guanines (Gu) can capture adenine
(Ad) tightly and then two guanines (Gu) and one adenine (Ad) form a
tripolymer layer structure. With the help of π-π stacking bonding, the
adenine (Ad) can be near to Eu3+ ion in EuIII-dtpa-bis(guanine) com-
plex and form coordination bond. Moreover, the formation of π-π stack-
ing binding makes the nitrogen atom (or amino group) of adenine (Ad)
further be closed to the Eu3+ ion until forming a stable and complete co-
ordination bond. At the same time, the original coordinate water mole-
cule will be replaced by the closer adenine (Ad). Under normal
conditions, the nitrogen atom is difficult to replace the oxygen atom.
Nevertheless, due to the presence of π-π stacking bonding the adenine
(Ad) is forced to be closed to the Eu3+ ion in EuIII-dtpa-bis(guanine)
complex forming a new coordination bond. Because of the change of li-
gand field around the Eu3+ ion, the fluorescence intensity of EuIII-dtpa-
bis(guanine) solution is also changed obviously. That is, the fluores-
cence intensity was distinctly enhanced after adding the adenine (Ad).
In addition, only in the presence of adenine (Ad) the EuIII-dtpa-
bis(guanine) solution gives greater fluorescence intensity. In contrast,
tion): LOD = 3σ/s, the calculated LOD is about 4.70 × 10−7 mol L−1
.
It can be seen from Table 1 that the method proposed in this work
showed a relatively wide detection range and low detection limit com-
pared with those reported in literatures. Particularly, due to the high sen-
sitivity and specificity to adenine (Ad) and outstanding anti-interference
to other base compounds with similar molecular structures and chemical
compositions to adenine (Ad), the EuIII-dtpa-bis(guanine) complex can
be used as an excellent fluorescence probe to detect adenine (Ad).
3.6. Reaction Mechanism and Process of EuIII-dtpa-bis(guanine) With Ade-
nine (Ad)
As shown in Fig. 6, the binding mechanism and process of EuIII-dtpa-
bis(guanine) as fluorescence probe with adenine (Ad) was proposed. It
Table 1
Comparison of the proposed method with others.
Detection method
Linear range (×10−6 mol/L)
LOD (×10−6 mol/L)
R
Reference
Electrochemical luminescence
Electrochemistry
Electrochemistry
Electrochemistry
Electrochemistry
Electrochemistry
Fluorometry
Fluorometry
Fluorometry
Fluorometry
5.0–100
2.5–150
1–132
2–200
25–200
1.0–46
1.40
0.75
0.70
0.58
0.76
0.33
0.08
0.53
0.019
0.47
0.9980
0.9993
–
0.9956
0.9980
0.9889
0.9993
0.9926
0.9991
0.9758
[4]
[10]
[14]
[15]
[16]
[17]
[21]
[22]
0.6–90
1.3–66
4.96 × 10−2–1.09
5.0–50
[23]
This work

F. Tian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179 (2017) 194–200
199
Fig. 6. The structure of Eu-dtpa-bis(guanine) + Ad (adenine) and reaction process of Eu-dtpa-bis(guanine) and adenine (Ad).
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upon the addition of other base compounds including guanine (Gu), hy-
poxanthine (Hy), xanthine (Xa) and uric acid (Ur) to the EuIII-dtpa-
bis(guanine) solution, the fluorescence emission intensity was slightly
quenched. Thus, it was confirmed that the sensitivity and specificity of
EuIII-dtpa-bis(guanine) complex as a fluorescence probe to detect ade-
nine (Ad).
4. Conclusion
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In this presented work, the adenine fluorescence probe (EuIII-dtpa-
bis(guanine)) was successfully designed and synthesized based on
Eu(III) complex with new dtpa-bis(guanine) ligand. In the EuIII-dtpa-
bis(guanine) complex, two guanine molecules whose composition and
structure are similar to that of adenine at the two ends (up and
down), can combine with adenine exclusively with the help of π-π
stacking interaction between adenine and guanine. Due to the forma-
tion of coordination bond of adenine with Eu(III) ion in the EuIII-dtpa-
bis(guanine) complex, the fluorescence intensity was enhanced clearly.
The addition of other bases such as guanine, xanthine, hypoxanthine
and uric acid to the EuIII-dtpa-bis(guanine) solution does not obviously
change the fluorescence emission intensity. Therefore, it was confirmed
that the EuIII-dtpa-bis(guanine) as a fluorescence probe has sensitivity
and specificity in the detection of adenine. Furthermore, a linear re-
sponse of fluorescence emission intensity of EuIII-dtpa-bis(guanine) at
570 nm as a function of adenine concentration was observed in the
range of 0.00–5.00 × 10−5 mol L−1. According to the formula of LOD
(limit of detection), the calculated limit of detection is about
4.70 × 10−7 mol L−1
.
Acknowledgments
The authors greatly acknowledge the National Science Foundation of
China (21371084 and 31570154), Key Laboratory Basic Research Foun-
dation of Liaoning Provincial Education Department (L2015043), Liao-
ning Provincial Department of Education Innovation Team Projects
(LT2015012) and Youth Science Foundation of Liaoning University
(2013LDQN14) for financial support. The authors also thank our col-
leagues and other students for their participating in this work.
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