Water-soluble Glucosamine-coated AIE-Active Fluorescent Organic Nanoparticles: Design, Synthesis and Assembly for Specific Detection of Heparin Based on Carbohydrate–Carbohydrate Interactions

Article abstract of DOI:10.1002/asia.201901153

Two water-soluble carbohydrate-coated AIE-activate fluorescent organic nanoparticles TPE3G and TPE4G were designed and synthesized for the detection of heparin. Different from the reported strategy, we not only utilized the general detection mechanism of

Full text of DOI:10.1002/asia.201901153

DOI: 10.1002/asia.201901153
Communication
Water-soluble Glucosamine-coated AIE-Active Fluorescent Organic
Nanoparticles: Design, Synthesis and Assembly for Specific
Detection of Heparin Based on Carbohydrate–Carbohydrate
Interactions
Yan-ming Ji, Guang-jian Liu, Cui-yun Li, Yi-chen Liu, Min Hou, and Guo-wen Xing*[a]
tivated clotting time assay (ACT), antifactor Xa activity assay,
electrochemical method and protamine binding method,[5]
Abstract: Two water-soluble carbohydrate-coated AIE-acti-
vate fluorescent organic nanoparticles TPE3G and TPE4G
were designed and synthesized for the detection of hepa-
rin. Different from the reported strategy, we not only uti-
lized the general detection mechanism of electrostatic in-
teractions, but also introduced the concept of carbohy-
drate-carbohydrate interactions (CCIs) to enrich the detec-
tion mechanism of heparin. TPE3G can serve as an effi-
cient “turn-on” probe with higher selectivity towards
heparin than TPE4G. TEM studies revealed that the micro-
aggregated TPE3G was encapsulated with the heparin
chain to form a complex self-assemblied composite and
emits strong fluorescence. It is believed that the results il-
lustrated in this study provide a novel strategy based on
CCls to design water-soluble and more efficient bio-
probes for various biological and clinical applications.
have some shortcomings, such as time-consuming, lack of in-
ternational standards, being susceptible to interference from
many factors and poor consistency of treatment scope. There-
fore, it is still an urgent need to develop a new strategy that
can monitor heparin level sensitively and rapidly in the clinic
applications.
In recent years, the studies on aggregation-induced emission
(AIE) have developed rapidly since it was discovered in 2001,
which has drawn great attention to fabricate various bio-
probes for a wide applications in biological detection and ma-
terial science.[6–8] Especially, functionalized AIE-active fluores-
cent organic nanoparticles (FONs) have been developed as bio-
compatible luminescent nonmaterials for cell imaging and bio-
medical applications due to their unique characteristics,
among which several kinds of heparin-specific AIE-active FONs
have been reported. For example, Li and co-workers[9] prepared
AIE-active nanofibers to detect serum heparin. Due to the elec-
trostatic interactions and static quenching, the fibers showed
the ratiometric color changes from yellow to cyan. However,
the water solubility of the fibers was poor, and the detection
was carried out in a mixture of DMSO and water. Zhang’s
group[10] and Tong’s group[11] reported a ratiometric fluores-
cence detection strategy based on AIE-active FONs for heparin
through electrostatic interactions with low detection limit,
however, the FONs used in the method still had defects in
poor water solubility and low sensing selectivity. In addition,
different tetraphenylethylene (TPE) based “turn-on”fluorescent
nanoparticles[1b,12–15] decorating with amine/ammonium or
other positively charged residues as the recognition groups
were designed and synthesized for the detection of heparin.
These probes have low detection limits, but they all have the
problems of poor water solubility and narrow linearity range.
At present, the detection mechanism for heparin with AIE-
active FONs as probes is mostly the strong electrostatic inter-
actions between positive charged AIE probes and heparin with
sulfonic groups.
Heparin (Hep), first named from the liver, is a linear and highly
sulfated glycosaminoglycan (GAG) that is strongly acidic and
has
a high density of negative charges, as shown in
Scheme 1.[1] Heparin is also found in tissues such as lungs, vas-
cular walls, and intestinal mucosa, which is a natural anticoa-
gulant substance in mammals. The physiological heparin levels
in human plasma range from
1
to 2.4 mgmLÀ1 (1.76–
4.24 mm),[1d] and the therapeutic dosages of heparin are sug-
gested to be 0.2–0.8 UmLÀ1 (2.82–11.29 mm) during clinical sur-
gery as anticoagulant reagent.[1e] It plays an important role in
regulating various physiological functions, including cell
growth and differentiation, immune system problems, coagula-
tion, as well as lipid transport and metabolism.[2] In clinic, hep-
arin can also be used for the treatment of thrombosis, hemo-
dialysis, cardiovascular surgery and many other surgical opera-
tions.[3] Like many medicines, excessive heparin levels can
cause many problems, such as bleeding, thrombocytopenia
and hyperkalemia.[4] Traditional heparin detection methods, in-
cluding activated partial thromboplastin time assay (APTT), ac-
With this in mind, the carbohydrate-carbohydrate interac-
tions (CCIs) have been considered as the crucial mediators for
cellular signal transduction including cell recognition and ad-
hesion etc.[16,17] Based on the literature reports,[18,19] we con-
cluded that CCIs mainly include: i) intermolecular hydrogen
binding, ii) intramolecular hydrogen binding, iii) intertwining
and self-assembling of carbohydrate chains. However, as a kind
of important polysaccharide, there were no report that heparin
[a] Y.-m. Ji, Dr. G.-j. Liu, C.-y. Li, Y.-c. Liu, M. Hou, Prof. G.-w. Xing
College of Chemistry
Beijing Normal University
Beijing 100875 (P. R. China)
E-mail: gwxing@bnu.edu.cn
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/asia.201901153.
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Scheme 1. The chemical structures of three GAGs.
was detected based on carbohydrate-carbohydrate interactions
to fabricate AIE-active probes.
TPE3G/TPE4G and the corresponding intermediates were de-
1
termined by H/13C NMR and HRMS to confirm the high purity
Herein, we designed and synthesized two water-soluble AIE-
active carbohydrate coated TPE molecules, named TPE3G and
TPE4G, respectively according to the amount of glucosamine
residues on them (Scheme 2). The purpose to use glucosamine
as the carbohydrate moiety of the target molecule is that glu-
cosamine possesses two crucial factors, one is highly hydro-
philic to make the functionalized TPE FONs water-soluble, the
other is that there are hydroxyl and amino groups in the sugar
structure which can served as the positions of carbohydrate-
carbohydrate interactions and electrostatic interactions. As
mentioned above, heparin is a kind of macromolecular poly-
mer with high density of negative charges, while the amino
groups in TPE3G and TPE4G could form ammonium cations in
physiological buffer solution (pH 7.4). The probes and heparin
could be strongly combined through the electrostatic interac-
tions and carbohydrate-carbohydrate interactions. It was ex-
pected that TPE3G/TPE4G-heparin composites could be highly
aggregated and emit strong fluorescence by the AIE effect of
TPE. The reasons of designing two different structures of mole-
cule TPE3G/TPE4G were to explore the distinction of sensitivi-
ty, detection limit, linear range for heparin detection under the
premise of different distribution positions of carbohydrates, as
well as the self-assembly difference between the two probes
and heparin. Since one side of TPE3G was hydrophobic (TPE)
and the other side was hydrophilic (glucosamine), the amphi-
philic molecule TPE3G could be self-assemble to form water-
soluble AIE-active fluorescent organic nanoparticles. As a com-
parison, the middle part of TPE4G was hydrophobic and the
four corners were hydrophilic. Therefore, the assembly state of
TPE4G and heparin should be different from that of TPE3G. In
this work, we deeply investigated the AIE properties of TPE3G
and TPE4G, and found that TPE3G was an efficient water-solu-
ble AIE-active FONs to specifically discriminate heparin from its
interferential species chondroitin sulfate (Chs) and hyaluronic
acid (HA), which have very similar structures with each other
(Scheme 1).
of the target products for the next fluorescence detection
studies.
As expected, both TPE3G and TPE4G have excellent water
solubility, and their fluorescence spectrum were observed at
different concentrations in HEPES buffer solution (pH 7.4). Fig-
ure 1(a) and (b) show that there were multiple linear ranges
for both probes, and the abscissa of the intersection point of
each two lines was called critical micelle concentration (CMC),
indicating the concentration values of TPE3G and TPE4G
when forming micelles. The results suggested that the amphi-
philic TPE3G and TPE4G should self-assemble in aqueous solu-
tion to form fluorescent organic nanoparticles. When the con-
centration of TPE3G was lower than 3.33 mm and the concen-
tration of TPE4G was lower than 2.82 mm, the background fluo-
rescence intensity was much weaker, and the probes should
not be aggregated obviously. Considering the comparability of
heparin response on the two probes, the concentration of
1 mm was selected for the following experiments.
Next, we measured the response curves of TPE3G and
TPE4G upon different heparin concentrations, respectively. As
shown in Figure 1(c) and (d), the results showed that the emis-
sion intensity of TPE3G enhanced gradually with increasing
the heparin concentrations, and the response signals remained
stable after reaching a certain concentration (about 7.2 mm). In
order to eliminate the distinctions of different initial emission
intensities that could occur during the measurement, the rela-
tive growth value [(F-F0)/F0] was used to analyze the concentra-
tion trends. TPE3G shows a linear trend in the range of 0–
7.2 mm with a slope of 0.38251 LmmolÀ1, and the standard
error of the slope was 0.01003 LmmolÀ1 (Figure 1(c)). The de-
tection limit based on the 3s=S method (LOD) was calculated
to be 0.2198 mm, where s was the standard deviation of fluo-
rescence intensity for 11 blank controls, 3 was the 3 times
signal-to-noise ratio, and S was the slope of linear trend. Nota-
bly, TPE3G afforded excellent water solubility, good detection
limit and the broadest linear range among the similar TPE
probes of heparin detection (Table S1). However, the emission
intensity of TPE4G showed a logarithmic growth trend with
the increasement of heparin concentrations (Figure 1(d)). Since
the above LOD calculation method is only applicable to graphs
with linear relationship, the detection limit cannot be calculat-
ed for TPE4G at this time. The insets of fluorescence photo-
graphs show the fluorescence changes in absence (left) or
presence (right) of heparin with the two probes. As can be
seen from the photographs, TPE3G solution changed the color
As indicated in Scheme 2, the carbohydrate hydrophilic
moiety 5 (azido derivatives of glucosamine) was synthesized
from glucosamine through 4-step protective group manipula-
tion reactions. TPE3G was prepared via double successive Cu+
-catalyzed click reactions with 5, AIEgen 6 and tetraalkyne 7 as
substrates followed by the global deprotection reactions. Simi-
larly, TPE alkyne 10 was reacted with 4 equivalents of glucosa-
mine derivative 5 to afford 11, which was further removed the
protective groups to give TPE4G. The chemical structure of
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Scheme 2. Synthetic routes of TPE3G and TPE4G.
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Figure 1. Plots of fluorescence intensity at 500 nm at different concentrations of TPE3G (a) and TPE4G (b) in HEPES buffer solution (10 mm, pH 7.4); Insets: PL
spectrum of corresponding plots. Plots of fluorescence relative value at 500 nm of TPE3G (c) and TPE4G (d) in HEPES buffer solution (10 mm, pH 7.4) with in-
creasing concentration of heparin, [TPE3G/TPE4G]=1 mm; Insets: PL spectrum of corresponding plots and the photographs of TPE3G/TPE4G in absence (left)
or presence (right) of heparin. lex(TPE3G) =333 nm; lex(TPE4G) =343 nm.
from almost colorless to cyan, and TPE4G changed the color
from dark green to bright green under Ultra-Violet lamp
(365 nm). These phenomena could also indicate that the re-
sponse of TPE3G and TPE4G to heparin both belong to the
“turn-on”probes.
fluorescent organic nanoparticles used for sensing heparin
than TPE4G.
In order to further clarify the binding mode of TPE3G or
TPE4G with heparin, the transmission electron microscope
(TEM) imaging of TPE3G/TPE4G and heparin themselves (Fig-
ure 3(a)/Figure S1, Figure 3(b)), as well as the composites of
TPE3G and TPE4G binding to heparin (Figure 3(g), Figure S2)
were obtained. Figure 3(a) was the image of TPE3G which was
self-assembled, with an average diameter of about 30 nm. The
size distribution of TPE3G (70 nm) in aqueous solution was de-
termined by dynamic light scattering (DLS) technique (Fig-
ure 3(c)), which was higher than that obtained by TEM, since
the data provided by DLS was the hydrodynamic particle size.
Figure 3(d) and (e) are the DLS histograms of heparin and
TPE3G binding to heparin, respectively. The average hydrody-
namic diameter of TPE3G binding to heparin (1700 nm) was
slightly higher than that of heparin (1300 nm), which basically
met the experimental facts after the combination of the two
species. We also obtained the photographs of the Tyndall
effect of TPE3G and TPE3G binding to heparin (Figure 3(f)),
which further supported the fact that they formed large hydro-
dynamic particle size micelles. Figure 3(g) are TEM images of
TPE3G binding to heparin. It can be observed from the inset
that the composite was thick in the middle and thin at the
edges, suggesting that it could form tubular or spherical struc-
tures. To further characterize the structure, scanning transmis-
To our delight, Figure 2(a) shows TPE3G could efficiently dis-
criminate the main interferents Chs and HA from heparin. Chs
and HA have similar carbon chain structures with heparin, but
their density of negative charges are lower than that of hepa-
rin. Upon adding Chs or HA (100 mm) into TPE3G solution, no
emission intensity enhancement was observed. However, with
the same treatment of heparin, the fluorescence intensity of
TPE3G increased significantly. The results indicated that the
weaker binding of Chs or HA with TPE3G, due to its lower neg-
ative charge density, should attribute to the no fluorescent re-
sponse. In addition, the fluorescence intensity of TPE3G was
measured with other possible interferents, including ATP and
BSA that contain high density negative charges, small anions
such as Na2HPO4, NaAc and citric acid, as well as other small
molecule carbohydrate compounds. The experimental results
showed that none of these substances could give positive fluo-
rescent signals to TPE3G. Interestingly, TPE4G gave fluorescent
response to a certain degree to Chs and BSA, which was about
1/2 to 1/3 value of heparin. Except for Chs, BSA and Hep,
TPE4G had weak response to other substances (Figure 2(b)).
Above findings illustrated that TPE3G was a better AIE-active
Figure 2. Selectivity detection histograms of heparin by TPE3G (a) and TPE4G (b) at 500 nm in HEPES buffer solution (10 mm, pH 7.4); Insets: PL spectrum of
corresponding histograms. The concentration of heparin was 50 mm, BSA was 1 mgmLÀ1, and others were 100 mm; lex(TPE3G) =333 nm; lex(TPE4G) =343 nm.
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Figure 3. TEM images of TPE3G (a) and heparin (b). DLS histograms of TPE3G (c), heparin (d) and the composites of TPE3G binding to heparin (e) show the
distribution of particle size. The colorimetric cups (f) show the Tyndall effect of TPE3G in absence (left) or presence (right) of heparin. (g) TEM images of the
composites of TPE3G binding to heparin; Inset was the enlargement image of a part. STEM (h) and EDS mapping (i) images of the inset in Figure 3(g). The el-
ements intensity profile line graph (j) of the composites shown in Figure 3(i). 5 mm of TPE3G/TPE4G and 50 mm heparin were used in the TEM studies.
sion electron microscopy (STEM) (Figure 3(h)) and energy dis-
persive spectrometer (EDS) mapping (Figure 3(i), Figure S3)
was performed for this area successively. In addition, an ele-
ments intensity profile was made according to the arrow direc-
tion in Figure 3(i) (from outside to inside) to obtain a series of
elements content broken line graphs (Figure 3(j)). As shown in
the graph, the sodium element content was the highest, the
nitrogen and sulfur elements contents were low, the carbon
and oxygen elements contents were moderate, and the chlor-
ine element was basically located on the surface of the compo-
sites. The contents of the six elements all gradually decreased
from outside to inside, but the carbon and oxygen elements
decreased less, and the nitrogen and sulfur elements were
close to zero inside of the composites. Since heparin contains
carbon, nitrogen, oxygen, sulfur, sodium and other elements,
but TPE3G contains carbon, nitrogen and oxygen elements,
we speculated that after the combination of the two, a struc-
ture was formed with TPE3G inside (TPE inward, carbohydrate
outward) and heparin outside. The glucosamine derivatives
and heparin were combined through carbohydrate-carbohy-
drate interactions and electrostatic interactions.
combined with heparin to form more complex structures than
TPE3G.
In order to understand the self-assembly mechanism of
TPE3G with heparin more intuitively, we draw a hypothetical
structure illustration to depict its shape more vividly in
Scheme 3. After being dissolved in aqueous solution, TPE3G
self-assembled into an internally hydrophobic, externally hy-
drophilic spherical nanoparticles due to its amphiphilicity. At
this time, the TPE micro-aggregated and emitted weak back-
ground fluorescence. Then, the spherical composites combined
with heparin to form long chain composites, and at this point,
the composites could emit strong fluorescence. Finally, multi-
ple long chains intertwined to form what we eventually ob-
served in TEM images. Notably, based on the images taken by
TEM, we speculated the self-assembly situation of TPE4G and
heparin, should be different from that of TPE3G. The chemical
structure of TPE4G is shaped like the letter “X”, with its four
corner glucosamines, it can form a kind of reticular structure
with heparin by carbohydrate-carbohydrate interactions and
electrostatic interactions. In addition, the formed reticular
structures were not limited to bind to one heparin chain.
Therefore, the TPE4G-heparin composites’ structure was cross-
linked and tridimensional. Due to the more complexities of
TPE4G-heparin than TPE3G-heparin, TPE4G had a low specific-
ity during the detection of heparin compared with the interfer-
ential species Chs and HA.
As for TPE4G, it was certain that TPE4G and heparin could
combine by the interactions of electrostatic force and carbohy-
drate-carbohydrate interactions to make TPE aggregated and
emitted strong fluorescence. Figure S2 and Figure S4 showed
the TEM, STEM and EDS mapping images of the composites of
TPE4G binding to heparin. As can be seen from the STEM
image (Figure S2c), it seemed that heparin and TPE4G
were wrapped and intertwined with each other, with some re-
ticular structures and holes in them. Therefore, TPE4G was
In a summary, we combined tetraphenylethylene, a simple
AIEgen, and glucosamine, a common carbohydrate to fabricate
two novel water-soluble AIE-active fluorescent organic nano-
particles TPE3G and TPE4G, which were used to detect hepa-
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Scheme 3. The self-assembly mechanism illustration of TPE3G binding to heparin.
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It is found that TPE3G was better than TPE4G for specifically
sensing heparin with good detection performance. The detec-
tion limit of TPE3G for heparin was 0.2198 mm, and the linear
range was 0–7.2 mm, which were basically superior to most of
other reported water-soluble TPE probes for heparin detection.
More importantly, TPE3G can specifically discriminate heparin
from its interferential species Chs and HA. TEM and DLS were
utilized to clarify the self-assembly mechanism of TPE3G with
heparin. The carbohydrate-carbohydrate interactions and elec-
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and sensing process between TPE3G and heparin. It is be-
lieved that the results illustrated in this study provide a novel
strategy based on CCls to design water-soluble and more effi-
cient bio-probes for various biological and clinical applications.
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Keywords: aggregation-induced emission · assembly
carbohydrate-carbohydrate interactions heparin
nanoparticles · tetraphenylethylene
·
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Manuscript received: August 19, 2019
Revised manuscript received: September 4, 2019
Accepted manuscript online: September 5, 2019
Version of record online: September 16, 2019
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