A cationic on-off fluorescent sensor with AIE properties for heparin and protamine detection

Article abstract of DOI:10.1039/d1nj02659c

In this research, a distyryl-anthracene derivative (DSAI) with two quaternary ammonium groups was synthesized. It emitted strong yellow-green fluorescence after aggregating with heparin through electrostatic attraction. In the presence of protamine, the fluorescence of DSAI remarkably quenched because of the greater affinity between heparin and protamine. Based on the change of fluorescence signal, a highly sensitive and selective method is successfully constructed to detect heparin and protamine. The DSAI sensor toward heparin showed a broad linear range (0.1 to 1.1 μg mL^?1) and a low detection limit (down to 5.64 ng mL^?1). The linear detection for protamine was in a wide range (0.1 to 1.7 μg mL^?1) with the detection limit of 5.20 ng mL^?1. Moreover, DSAI was successfully applied to detect heparin and protamine in complicated goat serum samples with satisfactory results. This simple and ultrasensitive biosensor has potential applications in diagnostics, therapeutics and biological research.

Full text of DOI:10.1039/d1nj02659c

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A cationic on–off fluorescent sensor with AIE
properties for heparin and protamine detection†
Cite this: DOI: 10.1039/d1nj02659c
a
b
a
a
a
Yingxi Zhou, Hongbo Jiang, Yuting Wang, Song Zhao, Lianzhe Hu
and
a
Yan Zhang
*
In this research, a distyryl-anthracene derivative (DSAI) with two quaternary ammonium groups was
synthesized. It emitted strong yellow-green fluorescence after aggregating with heparin through
electrostatic attraction. In the presence of protamine, the fluorescence of DSAI remarkably quenched
because of the greater affinity between heparin and protamine. Based on the change of fluorescence
signal, a highly sensitive and selective method is successfully constructed to detect heparin and
ꢀ
1
Received 31st May 2021,
Accepted 5th August 2021
protamine. The DSAI sensor toward heparin showed a broad linear range (0.1 to 1.1 mg mL ) and a low
ꢀ1
detection limit (down to 5.64 ng mL ). The linear detection for protamine was in a wide range (0.1 to
ꢀ1
ꢀ1
1
.7 mg mL ) with the detection limit of 5.20 ng mL . Moreover, DSAI was successfully applied to detect
DOI: 10.1039/d1nj02659c
heparin and protamine in complicated goat serum samples with satisfactory results. This simple and
ultrasensitive biosensor has potential applications in diagnostics, therapeutics and biological research.
rsc.li/njc
traditional assays for heparin quantification, such as the acti-
vated coagulation time (ACT) or the activated partial thrombo-
Introduction
Heparin (Hep), a naturally occurring linear polymer consisting plastin time (APTT), are indirect and inaccurate due to their
1
3,14,18
predominantly of 1–4 linked uronic acid and glucosamine lack of specificity and potential interference.
However,
subunits, is known as the densest negatively charged biological fluorescence detection is a burgeoning detection method,
macromolecule. It is a reagent for DNA decondensation and which has the characteristics of high sensitivity and selectivity,
one of the oldest biological drugs that has been widely used as easy operation, and real time detection. And during the detec-
an anticoagulant during clinical procedures and treatment of tion process, the detection of the substance is capable of
1
–4
thrombotic diseases.
The quantification of heparin is very being directly identified by the fluorescence sensor. However,
important in anticoagulation therapy. A series of side effects conventional fluorescent small molecule sensors tend to
can be caused by an overdose of heparin, such as haemorrhage undergo p–p accumulation in aqueous solutions, resulting in
5
–7
19,20
or thrombocytopenia.
Protamine is a vasodilator and posi- aggregation-caused quenching (ACQ) effects.
Molecules
tively charged peptide, which can bond with heparin alleviating with an aggregation-induced emission property (AIE), a com-
8
–10
emergency bleeding.
To reverse the effect of heparin, pro- pletely opposite property to ACQ, can be used in sensing
tamine strongly binds to the sulfonate group of the heparin applications by amplifying the signal and provide a new option
1
1
21,22
molecule, forming a stable neutral salt complex. However, an for fluorescence detection.
In addition to the common
2
3–26
overdose of protamine may produce life-threatening side tetraphenyl ethylene,
effects such as systemic hypotension, pulmonary vasoconstric- cular structures, such as silole, triphenylethene, and dis-
there are reports of some AIE mole-
2
7
28
1
2–15
29
tion or allergic reactions.
tyrylanthracene (DSA). Modifications on the basis of these
Protamine can be detected inadequately by the traditional compounds can interact directly with the substrate and then
method measuring UV-absorption at 280 nm, a standard aggregate to produce fluorescence amplification for detection.
method for the quantification of proteins, because it is Precisely because its sensing method is simple and direct, it
composed of aliphatic amino acids with the absence of aro- will reduce the interference to a large extent, thus making it
3
0
matic amino acids. The administration of protamine is empiri- possible to reach a lower detection limit. Moreover to the best
1
6,17
cally based on heparin analysis in clinical practice.
The of our knowledge, few researchers have reported that heparin
and protamine are detected in one detection system with a
3
1–33
detection limit in single-digit nanograms per milliliter.
Thus, it is meaningful to develop this sensor.
a
Chongqing Key Laboratory of Green Synthesis and Applications, College of
Chemistry, Chongqing Normal University, Chongqing, China
Bashu Secondary School, Chongqing, China
b
Herein, we report a simple method to detect heparin and
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/d1nj02659c protamine. A DSA derivative with two quaternary ammonium
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groups, 4,4 -(1E,1 E)-2,2 -(anthracene-9,10-diyl) bis(ethene-2,1- with trichloromethane, and then dried overnight in vacuo at 50 1C.
1
diyl) bis(N,N,N-trimethylbenzenaminium (DSAI), was synthe-
6
H NMR (300 MHz DMSO-d ) d (TMS, ppm): 8.41–8.33 (m, 6H),
sized. DSAI was combined with heparin due to electrostatic 8.38–8.33 (d, J = 16.5 Hz, 2H), 8.12–8.04 (m, 8H), 7.61–7.58 (m, 4H)
13
attraction and aggregated. Then the as-prepared DSAI was used 7.08–7.02 (d, J = 16.5 Hz, 2H), 3.68 (s, 18H); C NMR (DMSO-d6,
as a fluorescent indicator and heparin was used as protamine 75 MHz): d (TMS) 146.5, 138.5, 135.2, 132.1, 128.9, 128.0, 127.9,
recognition units to construct a DSAI/Hep complex probe 127.8, 126.1, 125.8, 120.8, 56.5; HRMS (ESI) 249.1514 (Calcd:
2+
38 2
for ‘‘turn-off’’ assay for protamine. The limit of detection of 249.1539 for C36H N ) (Fig. S1–S3, ESI†).
ꢀ1
ꢀ1
heparin and protamine was 5.64 ng mL and 5.20 ng mL
,
respectively. It also showed a good detection effect in goat
serum. This simple and ultrasensitive heparin biosensor has
potential uses in diagnostics, therapeutics and biological
research.
Analytical procedure
Procedure for the detection of heparin. Aliquots of 8 mL
volume of prepared DSAI solution were added to 1.5 mL
centrifuge tubes, and then various concentrations (0 to 84 mg mL
ꢀ
1
)
of heparin solution were added. The final volume of the
mixtures was maintained at 1.0 mL by phosphate buffer
Experimental
Reagents and chemicals
(0.2 M, pH 7.0). The resulting solutions were adequately mixed
and incubated for 15 min. The fluorescence of the mixtures was
measured at 408 nm excitation. All measurements were per-
formed at room temperature and there is no need for the
removal of oxygen or the protection of nitrogen.
Heparin (Hep) and protamine (Pro) were purchased from
Sigma-Aldrich (Missouri, USA) with average molecular weights
of 6117 and 4070, respectively. All chemicals were of analytical
grade and purchased from Aladdin (Beijing, China), J&K (Beijing,
China) unless stated otherwise. Prior to use, all solvents were
purified by fractional distillation.
Procedure for the detection of protamine. 8 mL methanol
ꢀ1
ꢀ1
solution of DSAI (0.1 mmol L ) was first added to a heparin
aqueous solution (0.12 mg mL , 100 mL) in 1.5 mL centrifuge
NMR spectra were recorded using a Bruker Avance 300
tubes, and then different amounts of protamine (from 0 to
(Rheinstetten, Germany). Mass spectra were recorded on an
ꢀ1
2
1
0 mg mL ) were added. The mixture solution was diluted to
Agilent 1100 LC-MS system (California, America). UV-Vis
absorption spectra were recorded using a UV-2550 UV-Vis
spectrophotometer (Kyoto, Japan). Fluorescence spectra were
measured using a PerkinElmer LS-55 spectrophotometer (Mas-
sachusetts, America). HRMS was recorded using a Bruker
ultraflextreme MALDI-TOF system (Rheinstetten, Germany).
.0 mL with phosphate buffer (0.2 M, pH = 7.0), sonicated for
more than 5 minutes and mixed thoroughly. Then their fluores-
cence measurement was performed under excitation at 408 nm.
Selectivity to heparin and protamine. 8 mL methanol solution
of DSAI and phosphate buffer solution (0.2 M, pH = 7.0) were
added to a 1.5 mL centrifuge tube. They were mixed into a solution
with a final volume of 1 mL and pH 7.0, and then the fluorescence
Synthesis of DSAI
intensity was measured. Different substances with the same
0
0
0
ꢀ1
Synthesis of compound 1 (4,4 -(1E,1 E)-2,2 -(anthracene- concentration of 10 mg mL , sodium phosphate (Na
2
HPO
4
),
9
,10-diyl) bis(ethene-2,1-diyl) bis(N,N-dimethylaniline)). Tetra- adenosine triphosphate (ATP), sodium citrate, vitamin C, hyaluro-
ethyl anthracene-9,10-bis (methylene)phosphonate (2.40 g, nic acid (HA), sodium cholate hydrate, bovine serum albumin
.00 mmol), which was synthesized according to known (BSA), and heparin (Hep), were added to the DSAI phosphate
5
procedures, was stirred with t-BuOK (1.68 g, 15.00 mmol) in buffer solutions separately under the same conditions above. The
THF (50 mL) under nitrogen. Compound 4-(dimethylamino) total volume was kept at 1 mL, and their fluorescence intensity
benzaldehyde (1.79 g, 12.00 mmol) in THF (50 mL) was added was measured.
ꢀ
1
to the solution that was kept in an ice bath and the mixture was
8 mL methanol DSAI and 100 mL heparin (12 mg mL ) were
stirred for 18 h at room temperature. The resultant precipitate mixed with 0.2 M phosphate buffer (pH = 7.0) to make a 1 mL
was washed successively with MeOH and filtered off to yield solution with pH 7.0, and the fluorescence intensity was
1
a yellow coloured compound. H NMR (300 MHz CDCl ) d measured. Under the same DSAI and heparin concentration
3
(
7
TMS, ppm): 8.44–8.41 (m, 4H), 7.75–7.69 (d, J = 16.5 Hz, 2H), conditions above, sodium chloride (NaCl), calcium chloride
.60–7.57 (d, J = 8.5 Hz, 4H), 7.45–7.42 (m, 4H), 6.87–6.82 (CaCl ), melamine, cysteine (Cys), glutathione (GSH), trypto-
d, J = 6.8 Hz, 2H), 6.83–6.80 (d, J = 3.3 Hz 4H), 3.03 (s, 12H); phan, and protamine (10 mg mL ) were mixed separately and
2
ꢀ1
(
1
3
C NMR (CDCl
27.6, 126.6, 126.1, 124.8, 120.7, 112.6, 40.5.
3
, 75 MHz): d (TMS) 150.5, 137.3, 133.0, 129.7, the total volume was kept at 1 mL.
Heparin detection in goat serum. For the assay of heparin in
Synthesis of DSAI (4,4 -(1E,1 E)-2,2 -(anthracene-9,10-diyl) goat serum samples, 30 mL of goat serum sample and 8 mL of
1
0
0
0
bis(ethene-2,1-diyl) bis(N,N,N-trimethylbenzenaminium)iodide). prepared DSAI were added to 1.5 mL centrifuge tubes, and the
An improved method based on the literature was used to prepare final volume of the mixtures was maintained at 1.0 mL. The
34
DSAI (Scheme S1, ESI†). Compound 1 (0.20 g, 0.43 mmol) was resulting mixture was adequately mixed and incubated at room
dissolved in 15 mL of trichloromethane, followed by the addition temperature for 15 min, and then the fluorescence of the
of 5 mL methyl iodide, and refluxed for 14 h under N
. After mixture was measured at 408 nm excitation. After that, the
2
cooling to room temperature, the mixture was filtered and washed goat serum samples with different concentrations of spiked
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heparin (0.36, 0.48, 0.60 and 0.72 mg mL ) were measured and the negatively charged sulfates or carboxylates in heparin.
under the same conditions. Then, the probe combined with heparin to form long chain
Protamine detection in goat serum. For the recovery composites, and at this point, the composites could emit strong
measurement, 30 mL of goat serum sample and 8 mL of DSAI fluorescence. The composites have become a new probe, DSAI/
were added to 1.5 mL centrifuge tubes. The final volume of the Hep complex. Due to the greater affinity between heparin and
mixture was maintained at 1.0 mL, and the mixture was reacted protamine, when protamine is added, DSAI is separated from
with ultrasound at room temperature for 5 min. Then the heparin and re-dispersed, resulting in a decrease in the fluores-
fluorescence of the mixture was measured at 408 nm excitation. cence intensity.
After that, the goat serum samples with different concentra-
The feasibility of the above-mentioned principle is proved by
the experiment. As shown in Fig. 1A, DSAI emitted dim fluores-
cence, but the fluorescence intensity increased remarkably after
adding heparin. An apparent fluorescence change is clearly
observed under irradiation at 365 nm (Fig. 1A, inset). When
protamine was added, an obvious fluorescence quenching
phenomenon appeared.
ꢀ
1
tions of spiked protamine (0.50, 0.70, 0.80, and 1.00 mg mL
)
were measured under the same conditions.
Results and discussion
Probe detection mechanism
The above changes are caused by the aggregation and
In recent years, AIE molecules have been used as fluorescent dispersion of DSAI with AIE properties. As described in
probes for protein detection, bioimaging, DNA analysis, and Fig. 1B, DSAI was dispersed in distilled water and the energy
biomolecular detection. When AIEgen is aggregated with of the excited state is dominated by non-radiative transition
biomolecules, the fluorescence quantum yield can be due to vibrations and rotations of molecules. The aggregation
improved. As a derivative of DSA with typical AIE properties, (greater than 100 nm) occurred after the interaction between
DSAI can produce an AIE effect to achieve the purpose of DSAI and heparin and the radiation channel is opened. The re-
detection.
dispersion of DSAI could be found from the DLS data and
The more detailed spectral characteristics of DSAI were quenched the fluorescence in the presence of protamine. The
measured. The absorption, excitation and emission of com- binding between the sensor and heparin and heparin and
pound 1 were measured in Fig. S4 (ESI†), which were at 405 nm, protamine is due to electrostatic attraction, which can be
430 nm, and 520 nm respectively. As shown in Fig S5 (ESI†), the proved by the potential changes after heparin and protamine
wavelengths of DSAI formed after interaction with methyl are added to DSAI. With the addition of heparin, the initially
iodide were measured to be 415 nm, 408 nm, and 519 nm, positive surface potential of DSAI gradually decreased and even
respectively. After being converted to a quaternary ammonium overturned to a negative potential (Fig. S6, ESI†). In contrast,
salt, its optical properties were not changed significantly, but it the addition of protamine recovered the potential to a positive
has been given a positive charge and an ability to electrostati- charge. This result illustrated that there are electrostatic inter-
cally bond. Furthermore, the remarkable AIE property of DSAI actions between the probe, heparin and protamine.
3
4
(
a tenfold increase in fluorescent strength) proves that DSAI
can be used as a sensor for detecting heparin.
The detection of heparin and protamine
The detection mechanism of heparin and protamine is
shown in Scheme 1. Due to the presence of two quaternary Heparin is directly recognized using DSAI and is also the
ammonium groups, DSAI has good solubility in methanol or a medium for subsequent testing. For obtaining the optimal
mixed solution of methanol/water and emits faint fluorescence. experimental conditions, the pH was optimized first. The
DSAI aggregated on heparin by electrostatic attraction between stronger fluorescence of DSAI could be observed when DSAI
the positively charged quaternary ammonium groups in DSAI methanol solution was added with heparin at the optimal
phosphate buffer solution (pH = 7.0), as shown in Fig. S7 (ESI†).
Moreover, the reaction time between DSAI and heparin
is optimized. As shown in Fig. S8 (ESI†), the fluorescence
Fig. 1 (A) Fluorescence spectra of (a) DSAI, (b) DSAI/HEP complex,
(
c) DSAI/Hep/Pro complex. [Inset: From left to right are DSAI, DSAI/hep,
Scheme 1 Schematic illustration of the heparin and protamine detection
mechanism for the fluorescence sensor.
and digital photos illuminated at 365 nm.] (B) DLS of DSAI in the presence
or absence of heparin and protamine (10 mg mL ).
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Fig. 2 (A) and (B) Fluorescence spectra (lex = 408 nm) of DSAI [0.1 mmol L
in phosphate buffer solution (0.2 M), pH = 7] in the presence of different
ꢀ1
amounts of heparin (from 0 to 84 mg mL ). [Inset: The relationship
between fluorescence intensity and the concentration of the heparin from
ꢀ
1
0
to 1.1 mg mL .].
intensity did not change after 15 minutes. Therefore, 15 min-
utes was chosen as the optimal reaction time for the following
experiments.
The fluorescence intensity change of DSAI with the increase
of the concentration of heparin is shown in Fig. 2A and B. The
Fig. 3 Fluorescence intensity of DSAI in 9 equivalents of some micro-
molecules and common less sulfated glycosaminoglycans.
fluorescence intensity increased with the increase of heparin
2
concentration. A linear plot (y = 98.4x + 88.86, R = 0.992) of the the fluorescence intensity decreases as the concentration of
fluorescence intensity at 519 nm was obtained, with a linear protamine increases (Fig. 4A and B). A splendid linearity of the
ꢀ
1
range of 0.1 to 1.1 mg mL and the detection limit reached fluorescence intensity at 519 nm (y = 568.21 – 71.1x) was fitted
ꢀ
1
ꢀ1
5
.64 ng mL . Compared to previously reported heparin in the concentration range of 0.1 to 1.7 mg mL , with a linear
2
fluorescence assays, the present method using DSAI as a correlation coefficient (R = 0.995). The detection limit for
fluorescent probe based on the electrostatic attraction can have heparin with this method could reach 5.20 ng mL . These
comparable or even lower detection limits (Table 1). measurement results clearly show that the DSAI/Hep complex
ꢀ1
We also investigated the response of DSAI to possible can be used as a more sensitive sensor for protamine compared
competitors. The turn-on responses of DSAI towards dibasic to previous reports (Table 1).
sodium phosphate (Na
2
HPO
4
), adenosine triphosphate (ATP),
To determine the selectivity of detection of protamine, the
sodium citrate, vitamin C, hyaluronic acid (HA), sodium cho- effect of interference from other potentially competing species
late hydrate, bovine serum albumin (BSA), blank and heparin including positively charged ions and related physiological
were evaluated. Interestingly, only heparin has a stronger molecules was investigated. For this purpose, the changes in
negative charge triggering the AIE response of DSAI (Fig. 3), the fluorescence intensity of the system upon the addition of
which suggests that the probe could selectively detect heparin. common biological species including sodium chloride (NaCl),
Protamine is a positively charged peptide showing a high calcium chloride (CaCl ), melamine, cysteine (Cys), glutathione
2
binding affinity for heparin. DSAI would be freed from the (GSH), tryptophan, and protamine were observed and recorded.
heparin after the addition of protamine and then turned off the The sharply fluorescent quenching in the system containing
AIE fluorescence. To keep the detection consistent, the pH of protamine demonstrated that the heparin–protamine complex
the buffer for protamine should be the same as the heparin was formed and DSAI redistributed in solution. In contrast,
detection. In order not to affect the result of protamine detec- when other competing species and molecules were used in the
tion by the mixing time, the fluorescence response at different system, almost no change in fluorescence intensity was
times was measured (Fig. S9, ESI†). When performing detec- observed (Fig. 5). As a result, a simple fluorescence sensing
tion, heparin was used as the identification unit of protamine, system is successfully developed for the rapid determination of
and its concentration was also very important. Fig. S10 (ESI†) protamine.
shows that the largest fluorescence quenching effect was at
The cyclability of this assay for detecting heparin and
ꢀ1
12.0 mg mL . After configuring the DSAI/Hep complex sensor, protamine is also important. For this purpose, quantitative
Table 1 List of different sensors to detect the performance of heparin and protamine
Serial number
Sensor
Heparin detection limit
Protamine detection limit
Ref.
ꢀ
1
ꢀ1
1
2
3
4
5
6
7
Pyrene-based ratiometric fluorescent sensor
Ratiometric scheme for the fluorescent detection
Accelerated etching of gold nanorods
AIE-based fluorescence probe
AIE-based fluorescence probe
DNA-templated gold nanoclusters
Perylene diimide derivative
153 ng mL
—
10 ng mL
37 ng mL
107.8 ng mL
35
36
37
38
39
40
41
ꢀ
1
7.7 ng mL
—
ꢀ
ꢀ
1
1
ꢀ1
ꢀ1
30 ng mL
47 ng mL
—
ꢀ
ꢀ
1
0.18 ng mL
1
0.15 mg mL
ꢀ
1
—
90 ng mL
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reduced. The corresponding results based on the four dupli-
cated measurements are displayed in Table S1 (ESI†). It could
be seen that the values measured using our proposed method
were consistent with the spiked values for the concentration of
heparin. These results indicate that this sensor has great
potential applications for the assay of heparin in real samples.
Moreover, to demonstrate the practical application of the
DSAI/Hep complex sensor for protamine, the detection of
protamine in the goat serum matrix was performed. The
ꢀ
1
Fig. 4 (A) and (B) Fluorescence spectra (lex = 408 nm) of DSAI [0.1 mmol L
ꢀ1
in phosphate buffer solution (0.2 M), pH = 7] and heparin (12.0 mg mL ) in the
ꢀ1
presence of different amounts of protamine (from 0 to 20 mg mL ). [Inset: determination results are illustrated in Table S2 (ESI†). As
The relationship between fluorescence intensity and the concentration of the can be seen, the recovery of protamine detected in serum
ꢀ1
protamine from 0 to 1.7 mg mL .].
samples ranged from 98% to 105%, which was satisfactory
for performing quantitative assays in biological samples.
Conclusion
In conclusion, a simple and highly sensitive fluorescent sensor
DSAI was synthesized. Based on its AIE property, the emission
intensity of DSAI could be enhanced by heparin and detection
of heparin was realized. The DSAI/Hep complex sensor imple-
mented highly sensitive detection of protamine because of the
stronger affinity between heparin and protamine. The limits of
detection for heparin and protamine both reached single-digit
nanograms per millilitre. Meanwhile, we also demonstrated the
analysis mechanism of DSAI for heparin and protamine detec-
tion. Furthermore, the as-developed DSAI sensor was also used
for the assay of heparin and protamine in goat serum samples
with satisfactory results. This work provides a simple, highly
sensitive and selective method for efficient sensing of heparin
and protamine.
Fig. 5 Fluorescence intensity of the DSAI/Hep complex in 7 equivalents of
different positively charged ions and physiological protein molecules.
heparin and protamine were alternately added into the solution
system to observe the increase and decrease of fluorescence.
Although the previous fluorescence intensity could not be fully
restored, significant changes in fluorescence intensity were
observed even after five cycles (Fig. S11, ESI†). This indicated
that this assay has the ability of reproducibility and
repeatability.
Conflicts of interest
There is no conflict to declare.
Acknowledgements
In addition, in a real sample, there would be an adequate This work is supported by the National Natural Science Foun-
concentration of heparin or protamine. Therefore, the determi- dation of China (No. 21604006) and the Youth Talent Devel-
nation of a high concentration of heparin and protamine is also opment Program of the Chongqing Normal University
meaningful. After increasing the concentration of the DSAI, the (No. 15XLB019).
response difference between the fluorescence intensity of
heparin at two high concentrations increased (Fig. S12, ESI†).
But unfortunately, a similar result was not observed in high
concentrations of protamine detection. This result revealed
that only a high concentration of heparin (without diluting)
could be detected by increasing the concentration of DSAI.
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