A novel water-soluble AIE-based fluorescence probe with red emission for the sensitive detection of heparin in aqueous solution and human serum samples

Article abstract of DOI:10.1016/j.tetlet.2017.08.023

Heparin is widely used to anticoagulation treatment in clinic, while the overdoses of heparin can cause potentially catastrophic complications. Thus, the selective and sensitive detection of heparin is of great importance. Herein, a novel water-soluble AIE-based fluorescent probe (TIBI) with red emission (650 nm) has been rationally developed to detect heparin by the electrostatic-interaction and ion replacing strategy. TIBI exhibited excellent selectivity and low detection limit (0.08 μM) for detection of heparin. Moreover, TIBI was successfully applied to detect heparin in complicated serum samples with satisfactory results. This study holds great promise for real time monitoring heparin in clinical application.

Full text of DOI:10.1016/j.tetlet.2017.08.023

Accepted Manuscript
A novel water-soluble AIE-based fluorescence probe with red emission for the
sensitive detection of heparin in aqueous solution and human serum samples
Shuqi Yang, Tang Gao, Jie Dong, Huan Xu, Feng Gao, Qian Chen, Yonghong
Gu, Wenbin Zeng
PII:
S0040-4039(17)31012-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.08.023
TETL 49213
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
7 July 2017
6 August 2017
8 August 2017
Please cite this article as: Yang, S., Gao, T., Dong, J., Xu, H., Gao, F., Chen, Q., Gu, Y., Zeng, W., A novel water-
soluble AIE-based fluorescence probe with red emission for the sensitive detection of heparin in aqueous solution
and human serum samples, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.08.023
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2
Graphical Abstract
Leave this area blank for abstract info.
A
novel
water-soluble
AIE-based
fluorescence probe with red emission for the
sensitive detection of heparin in aqueous
solution and human serum samples
1
1
1
2
2
2
2
1,*
Shuqi Yang , Tang Gao , Jie Dong , Huan Xu , Feng Gao , Qian Chen , Yonghong Gu , Wenbin Zeng

1
Tetrahedron Letters
A novel water-soluble AIE-based fluorescence probe with red emission for the
sensitive detection of heparin in aqueous solution and human serum samples
1
1
1
2
2
2
2
1,*
Shuqi Yang , Tang Gao , Jie Dong , Huan Xu , Feng Gao , Qian Chen , Yonghong Gu , Wenbin Zeng
a
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha 410013, China
The Third Xiangya Hospital, Central South University, Changsha, 410013, China
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Heparin is widely used to anticoagulation treatment in clinic, while the overdoses of heparin can
cause potentially catastrophic complications. Thus, the selective and sensitive detection of
heparin is of great importance. Herein, a novel water-soluble AIE-based fluorescent probe
(TIBI) with red emission (650 nm) has been rationally developed to detect heparin by the
electrostatic-interaction and ion replacing strategy. TIBI exhibited excellent selectivity and low
detection limit (0.08 μM) for detection of heparin. Moreover, TIBI was successfully applied to
detect heparin in complicated serum samples with satisfactory results. This study holds great
promise for real time monitoring heparin in clinical application.
Available online
eywords:
Heparin detection
Aggregation-induced emission
Long wavelength emission
Water-solubility
2
009 Elsevier Ltd. All rights reserved.
Human serum samples
Heparin
is
a
negatively
sulfated
polymeric
manipulation and high performance. Recently, some
10
glycosaminoglycan (GAG) with the highest charge density in
biological systems, primarily (>70%) consists of trisulfated
fluorescence probes, such as small molecule probes , peptide-
11 12
based probes , cationic conjugated polymers and quantum
1
13
disaccharide repeating units . Owing to its important role in
regulation of diverse biological processes such as cell growth,
differentiation, metabolism and inflammation , heparin is of
great importance in biological studies. It is also capable of
binding antithrombin III with high affinity, which powerfully
enhances antthrombin’s inhibition activity towards coagulation
dots have been developed to detect small molecules,
biomacromolecule, and microenvironment. However, some of
them are based on complicated nano-organic hybrid systems
with poor water-solubility and toxicity which limit their practical
2
14
applications . Meanwhile, the fluorescence emission of some
conventional fluorophores, such as coumarin, rhodamine,
benzothiazole derivatives and etc, was found to be often
partially or completely quenched in aqueous solution, and many
of these probes based on the formation of probe/ heparin
closely packed complexes, their fluorescence might suffer from
self-quenching because of the aggregation caused quenching
3
factors such as thrombin . So it has been widely used in both
anticoagulation treatment and venous thrombosis conditions
clinically. The prescribed therapeutic dosages of heparin are 2-8
-1
-1
U mL (17-67 μM) and 0.2-1.2 U mL (1.7-10 μM) during
cardiovascular surgery and postoperative long-term care,
4
15
respectively . Unfortunately, overdoses of heparin can cause
(ACQ) effect . Therefore, it is still highly demanded to explore
various potentially catastrophic complications, such as
fluorescent probes with good water-solubility and superior
luminescent property in aqueous solution for heparin detection.
haemorrhaging,
osteoporosis
and
heparin-induced
5
thrombocytopenia . Therefore, the selective and sensitive
quantification of heparin during surgery and postoperative
therapy are of critical importance.
In 2001, Tang and co-workers firstly reported the
1
6
phenomenon of "aggregation-induced emission" (AIE) . AIE
luminogens (AIE-gens) are almost non-luminescent when they
are dissolved in a good solvent but emit intensely in a poor
Up to now, several assays to monitor heparin have been
reported, including activated clotting time assay (ACT), activated
partial thromboplastin time assay (aPTT), capillary
17
solvent . Meanwhile, the AIE-gens have significant advantages,
such as bright luminescent in the aggregate state and large
6,7
18
electrophoresis and electrochemical methods . However, ACT
Stokes shift, etc . Recently, a variety of AIE-based fluorescent
8
and aPTT methods are not sufficiently reliable . On the other
probes have been developed for detection of many substances,
19
hand, the electrochemical assays suffer from some limitations,
such as low specificity and interference by other charged species
such as ions, organic small molecules and biomolecules, etc
.
However, the reported AIE-based fluorescent probes for heparin
often have short emit wavelength (< 600 nm), which may
interfere by the background fluorescence of endogenous
biomolecules. Thus it is highly desirable to design AIE-based
9
in serum . Thus, it is highly desirable to develop new methods
for detection of heparin with high accuracy and reliability. In
recent years, fluorescent assays have gained considerable
attention for monitoring analytes due to the low cost, easy

2
fluorescent probe for heparin detection with long wavelength
emission.
20
In previous studies , we discovered that the tetraphenyl
imidazole derivatives could act as a typical AIE gen, emitting
weak fluorescence in dispersed state and turning on its
fluorescence in aggregate state. In this work, we successfully
designed and synthesized a water-soluble AIE-based cationic
fluorescent probe TIBI for highly sensitive and selective
detection of heparin through an ion pairing mechanism. The
design rationale was illustrated in Fig 1. In our strategy, the
tetraphenyl imidazole group was treated as the AIE fluorophore,
Fig 2. (a) Fluorescence emission and (b)absorption spectra of 10μM
TIBI in DMSO/PBS mixtures with different PBS contents (λex = 425
and
a
2,3-dimethylbenzo[d]thiazol-3-ium
iodide
was
nm).
incorporated through a vinylene bridge to the poly-substituted
imidazole core, since the 2,3-dimethylbenzo[d]thiazol-3-ium
iodide not only could increase water solubility but also could
serve as an electron-withdrawing group to induce a strong
intramolecular charge transfer (ICT) transition and thus making
the emission wavelength shift to red obviously. The probe TIBI
exhibited weakly fluorescent in aqueous solution owing to the
free intramolecular rotation, when the probe was in the
presence of heparin, the electrostatic attractions between the
negatively charged groups of heparin with positively charged
TIBI would lead to the formation of TIBI/heparin complex,
emitting red bright fluorescence (650 nm) through the
restriction of intramolecular motion (RIM).
Fig 3. (a) Normalized fluorescence spectra of TIBI in different
solvents. Cyc, cyclohexane; THF, tetrahydrofuran; DCM,
dichloromethane; MeCN, acetonitrile; MeOH, methanol; DMF, N,N-
dimethylformamide; Concentration: 10 μM; λex = 425 nm; b)
Molecular orbital amplitude plots of HOMO and LUMO of TIBI
(Iodide ion was omitted, and the meaning of red and blue color is
the distribution of high-density electronic cloud.)
The synthesizing route of TIBI was shown in Scheme 1.
Firstly, the TIB was prepared as reported in the literature via
2
1
one-pot reaction method , then TIB was further reacted with
,3-dimethylbenzo[d]thiazol-3-ium iodide to generate the probe
2
TIBI in 52%yield. (see ESI for more details, Scheme S1-S3).
1
Molecular structure of the probe TIBI was confirmed by H
We then measured the fluorescence spectra of TIBI in
different water fractions. As shown in Fig 2a, when the water
fraction in the mixture was lower than 99%, TIBI was nearly
non-fluorescent. The phenomenon of non-fluorescent might be
due to the TIBI could be dissolved in DMSO solution easily, and
the good solubility of TIBI could make the molecule undergo
dynamic intramolecular rotations, reducing the luminescence.
However, when the fraction of water was higher than 99%, a
weak fluorescence signal in 650 nm was recorded (Ф=0.01). The
abnormal fluorescence emission suggested the generation of a
loose aggregation state. Unlike the dense aggregation states, the
loose aggregation state made the TIBI compounds have enough
free space to consume the radiative energy, resulting in nearly no
fluorescence. Meanwhile, owing to its heavy-atom effect, iodine
was a well-known effective fluorescent quencher, which could
help to accelerate the rate of intersystem crossing and hence
promoted the collision of the fluorophore, resulting in non-
1
3
NMR, C NMR and HR-MS analysis in the supporting
information (Fig. S2-S4).
Fig 1. Illustration of the proposed mechanism of interaction
between TIBI and heparin.
2
2
radiative relaxation . Based on the reasons as mentioned above,
the fluorescent signal of TIBI in aqueous solution was very
weak. Moreover, the UV absorption spectra of TIBI in different
water fractions were also measured. As shown in Fig 2b, in pure
DMSO solution, the absorption peak of TIBI located around 440
nm. Upon the water content increase to 99%, a significant blue
shift was observed and a new maximum absorption peak
appeared at 420 nm which attributed to the ICT transition of the
Scheme 1. Synthesizing route of TIBI
2
3
entire molecule and the production of H-aggregates . The
influence of ICT effect was proved by the fluorescence spectra of
TIBI in different solvents, As the solvent polarity increased (from
n-hexane to methanol), the emission bands of TIBM showed
gradual red-shifting from 575 nm to 710 nm (Fig. 3a). The results
indicated that the electron-donating tetraphenyl imidazole and the
electron-withdrawing 2,3-dimethylbenzo[d]thiazol-3-ium iodide
group offer an ICT process in the molecule. Moreover, the
influence of ICT effect was further proved via the computational

3
calculations. As shown in Fig 3b, in the highest occupied
molecular orbital (HOMO), the higher density of electron cloud
was distributed on electron-donating imidazole group, while in
the lowest unoccupied molecular orbital (LUMO), they tended to
be denser located around electron-withdrawing 2,3-
dimethylbenzo[d]thiazol-3-ium iodide unit.
As mentioned above, a weak fluorescence could be observed
when TIBI was dissolved in aqueous solutions. However, as
shown in Fig S1a, a bright red fluorescence “turn on” in 650 nm
was observed after the addition of heparin to a 10 μM TIBI PBS
solution (pH = 7.4, 10 mM) (Ф=0.16), which might attributed to
the fluorescence quenching iodide ion was availably displaced by
the negative charge of heparin, meanwhile, the aggregation of
TIBI driven by electrostatic interaction between the positively
charged 2,3-dimethylbenzo[d]thiazol-3-ium iodide and the
negatively charged heparin limited the free intramolecular
motion. As shown in Fig 4a,4b with an increasing amount of
added heparin, the fluorescence intensity of the TIBI at 650 nm
was enhanced gradually and could be directly observed by naked
eye under excitation of the UV lamp (λex = 365 nm). Analysis of
the fluorescence emission intensity data revealed that the
intensity increased linearly with the increasing concentrations of
heparin in the range of 0 -10 μM, which covered the clinical
dosage level (1.7-10 μM), and a good linear correlation
Fig 4. a) Fluorescence spectra of 10 μM TIBI in the presence of
different amounts of heparin (0-17 μM) in PBS buffer solution (pH =
7
.4, 10 mM) with λex= 425 nm; b) Fluorescence spectra of 10 μM
TIBI at 650nm in the presence of different amounts of heparin (0-17
μM) in PBS buffer solution (pH = 7.4, 10 mM) with λex= 425 nm.
Inset shows the photos of the solution of 10 μM TIBI in the absence
and presence of 8 μM heparin under illumination of portable light;
c)Plots of emission intensity in 650 nm against the concentration of
heparin with λex = 425 nm; d) Absorption spectra of 10μM TIBI in
the presence of different amounts of heparin (from 0 to17 μM) in
PBS buffer solution(pH = 7.4, 10 mM).
2
coefficient (R = 0.9906) can be obtained (Fig 4c). The calculated
detection limit for heparin with fluorescent methods based on
3
δ/s can reach 0.08 μM under this condition. Furthermore, as
shown in Fig S1b and Fig 4d, upon the addition of heparin in
TIBI solution, the UV-Vis absorption spectra of TIBI exhibited
a decrease in absorption intensity (at 420 nm) and a slight red-
shift in the absorption maximum(at 425 nm), which also proved
the formation of a new aggregation state.
The selectivity of TIBI for heparin was assessed by
measuring the fluorescence intensity in the presence of main
relevant species, including glucose, adenosine triphosphates
(
(
ATP), bovine serum albumin (BSA), cysteine, phosphate ion
Na PO ), chondroitin-4-sulfate (Chs) and hyaluronic acid (HA).
3
4
As shown in Fig 5, among these molecules, only the addition of
heparin in TIBI system resulted in an obvious increase in the
fluorescence emission intensity. It was worth pointing out that
the heparin analogues Chs and HA also did not produce
distinguished fluorescence enhancement when using TIBI as the
tested probe. These phenomena suggested that the probe TIBI
was highly selective for the detection of heparin, not suffering
from interferences from other ions and biomolecules. The high
selectivity of TIBI might attribute to the electrostatic interactions
between probe TIBI and heparin. Compared with other
molecules, heparin possessed more negatively charged groups, so
it had much stronger electrostatic interactions than other
molecules, and exhibited outstandingly affinity toward heparin.
Fig 5. Fluorescence intensities of TIBI (10 μM) upon addition of
heparin (4 μM) or other different ions and biomolecules (50 μM) (1.
Glucose, 2. ATP, 3. BSA, 4. Cysteine, 5. Na PO , 6. HA, 7. Chs, 8.
3
4
Heparin).
Fig 6. Time-dependent fluorescence intensity changes of 10 μM TIBI
in PBS buffer solution (pH = 7.4, 10 mM) at 650 nm upon addition of
varying concentrations of heparin with λex = 425 nm.

4
that heparin could interact with the positive charge of TIBI
through electrostatic interaction, and the formation of
nanoaggregates could restrict the intramolecular rotation
effectively, resulting in “turn-on” detection of heparin.
Based on the superior luminescent property of TIBI as
mentioned above, we further applied the probe TIBI for
detection of heparin in human serum samples to evaluate its
24
validity. According to the report's methods , the human serum
samples were analyzed using spiking standard heparin samples
at different concentrations (0, 2.5, 5.0 and 10.0 μM). The results
obtained were listed in Table 1, the measured recoveries of
heparin was ranged from 96 to 105% with less than 5.0% RSD
under the optimal conditions, which showed the probe TIBI has
the potential applicability to detect heparin in complex
biological systems.
Fig 7. DLS analyses the assemblies of TIBI (10 μM) in PBS buffer
solution (pH = 7.4, 10 mM) in the absence (a) and presence (c) of
heparin (8 μM). SEM analyses the assemblies of TIBI (10 μM) in PBS
buffer solution (pH = 7.4, 10 mM) in the presence (d) and absence
In conclusion, we have developed a simple, effective
fluorescent turn-on probe TIBI for detection of heparin based on
the synergistic strategy of AIE and electrostatic interaction. The
results indicated that probe TIBI exhibited good water-solubility,
excellent selectivity, very low detection limit (0.08 μM) and long
wavelength emission (650 nm). In addition, TIBI was successfully
used to detect heparin in human blood serum samples. The
superior detection ability made it a potential analytical tool for
detection of heparin in complex clinical samples.
(
b) of heparin (8 μM).
Table 1. Recovery results of real sample measurements
Sample
Added
Measured
Recovery
(%)
RSD
(
μM)
(μM)
(%,
n=3)
Acknowledgments
1
0%
0
Not found
2.42
-
-
The authors gratefully appreciate the support from the
National Natural Science Foundation of China (81671756). We
also acknowledge the Modern Analysis and Testing Center of
CSU for the NMR spectroscopic measurements.
diluted
human
serum
2
5
.5
.0
96.8
105.0
101.7
2.8
3.5
1.7
5.25
Supplementary Material
1
0.0
10.17
Supplementary data associated with this article can be found, in
the online version.
References and notes
The time-dependent fluorescent spectrum of probe TIBI
with heparin was further investigated. As shown in Fig 6, when a
low concentration of heparin (0.4 μM) was added, the emission
intensity of probe TIBI at 650 nm gradually increased and
reached the plateau at about 120 s. When the concentration of
heparin was increased to1.2 μM, the emission intensity of TIBI
could reach the maximum in 100s and remains stable in a long
time. These results showed that the probe TIBI could detect
heparin in a remarkably short time with high stability.
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6
Highlights
A novel water-soluble AIE-based fluorescence probe with red emission for the
sensitive detection of heparin in aqueous solution and human serum samples
1
1
1
2
2
2
2
1,*
Shuqi Yang , Tang Gao , Jie Dong , Huan Xu , Feng Gao , Qian Chen , Yonghong Gu , Wenbin Zeng
1
2
3
4
5
. An novel AIE-based compound TIBI was successfully designed and synthesized.
. TIBI exhibited great solubility in aqueous solution.
-1
. TIBI could detect heparin with very low detection limit (0.01U mL ).
. TIBI could exhibit red light emission (650 nm) while interacting with heparin.
. TIBI could be successfully applied to detect
heparin in human serum samples.