Fluorometric detection of lectin with water-soluble hyperbranched conjugated polymer using mannose mediation

Article abstract of DOI:10.1166/jnn.2012.5907

A water-soluble hyperbranched polymer containing boronic acid groups at the ends of the polymer, which are capable of binding to diol-containing mannose, was syntheized. The hyperbranched polymer was prepared by a palladium-catalyzed Suzuki cross-coupling reaction using the tribromo monomer for the hyperbranched type structure. The water-soluble hyperbranched polymer (HP) exhibited enhanced fluorescence intensity upon exposure to lectin in the presence of mannose compared to other proteins, such as lysozyme and cytochrome c, because mannose plays a key role in binding both lectin and HP resulting in selective sensing toward lectin. Copyright

Full text of DOI:10.1166/jnn.2012.5907

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Journal of
Nanoscience and Nanotechnology
Vol. 12, 4365–4369, 2012
Fluorometric Detection of Lectin with Water-Soluble
Hyperbranched Conjugated Polymer
Using Mannose Mediation
Daigeun Kim1, Geun Seok Jang2, Ji Hye Son2, Seongwon Seo2,
Jongho Kim3, Tae Hyeon Kim2ꢀ4, and Taek Seung Lee2ꢀ3ꢀ∗
1Organic and Optoelectronic Materials Laboratory, Department of Nanotechnology, Chungnam National University, Daejeon 305-764, Korea
2Organic and Optoelectronic Materials Laboratory, Department of Advanced Organic Materials and Textile System Engineering,
Chungnam National University, Daejeon 305-764, Korea
3Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 305-764, Korea
4Film Research Institute, Kolon Central Research Park, Kolon Industries, Inc., Gyeongbuk 730-030, Korea
A water-soluble hyperbranched polymer containing boronic acid groups at the ends of the polymer,
which are capable of binding to diol-containing mannose, was syntheized. The hyperbranched
polymer was prepared by a palladium-catalyzed Suzuki cross-coupling reaction using the tribromo
monomer for the hyperbranched type structure. The water-soluble hyperbranched polymer (HP)
exhibited enhanced fluorescence intensity upon exposure to lectin in the presence of mannose
compared to other proteins, such as lysozyme and cytochrome c, because mannose plays a key
role in binding both lectin and HP resulting in selective sensing toward lectin.
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Keywords: Fluorescence, Lectin Sensor, Water-Soluble Hyperbranched Polymer.
Copyright: American Scientific Publishers
1. INTRODUCTION
Boronic acids have been known for a few decades
for their ability to interact with diol-containing com-
pounds such as carbohydrates.11ꢁ12 They have been used
for the development of receptor and fluorescent probes for
sugars.13ꢁ14 The main advantage of using boronic acid as
a ligand group for sugars are the fast and reversible inter-
action with sugars.15ꢁ16
Carbohydrate-protein interactions, which play a role
in the interactions of proteins, viruses, and bacteria, are
among the most important events or mechanisms in bio-
logical systems.17ꢁ18 Lectins are carbohydrate-binding pro-
teins that mediate important biological processes such
as cell growth, the inflammatory response and viral
infections.19–21 A range of sensors for lectins have been
developed using different principles in the sensor design.
Among them, fluorescence-based lectin sensors employ
a sugar-containing probe molecule. In the present study,
water-soluble conjugated polymer containing mannose
group to use as a lectin sensor was designed and
synthesized.22
Recently, conjugated polymers have attracted considerable
interest because of their interesting properties derived from
a ꢀ-conjugated system.1–4 Linear conjugated ones, such as
poly(p-phenylenevinylene), polyfluorene, and their deriva-
tives, are the most extensively studied conjugated poly-
mers. However, there are some drawbacks that limit their
potential applicability, such as low solubility and high
viscosity.5ꢁ6
To solve these problems, several attempts have been
made to utilize longer and branched side chains or bulky
substituents, copolymerization techniques, and dendrimer
attachments.7ꢁ8 Among them, the hyperbranched system
has advantage over its linear counterpart in terms of
high solubility, good processability, and fewer unfavorable
intermolecular interactions and crystallization.9 In addi-
tion, it is possible to endow these hyperbranched archi-
tectures with functions such as enhanced adhesion, energy
harvesting and optoelectronic characteristics by controlling
the nature of the end groups.10
This paper reports the first water-soluble hyperbranched
polymer containing boronic acid, which shows high
binding properties with mannose. The number of mannose-
binding ligands, boronic acid, was maximized through the
synthesis of a hyperbranched polymer structure, which has
∗Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2012, Vol. 12, No. 5
1533-4880/2012/12/4365/005
doi:10.1166/jnn.2012.5907
4365

Fluorometric Detection of Lectin with Water-Soluble Hyperbranched Conjugated polymer Using Mannose Mediation
Kim et al.
0.4
150
comparable number of polymer end groups. The mannose-
mediated hyperbranched polymer synthesized has high
selectivity to lectin.
0.3
100
50
0
0.2
2. EXPERIMENTAL DETAILS
2.1. Instrumentation
0.1
The 1H NMR spectra were obtained on a Bruker DRX-300
spectrometer (Korea Basic Science Institute). Elemental
analyses were performed using a CE Instruments EA-
0.0
300
350
400
450
500
550
600
Wavelength [nm]
1110 elemental analyzer. The FT-IR spectra were obtained
using a Mattson Genesis II spectrometer. UV-vis absorp-
tion spectroscopy was carried out using a PerkinElmer
Lambda 35 spectrometer. The photoluminescence spectra
were obtained using a Varian Cary Eclipse equipped with
a xenon lamp excitation source.
Fig. 1. Absorption and emission spectra of HP (excited at 352 nm) in
aqueous solution; [HP] = 1ꢄ0×10−5 M for absorption spectra.
in water. However, the product precipitated was insol-
uble in water due mainly to its crosslinked structure
resulting from trifunctional monomer. Therefore, precise
control of the reaction time and tribromo monomer, tris(4-
bromophenyl)amine content is essential for obtaining a
water-soluble hyperbranched polymer. HP was character-
ized by 1H NMR, IR, and elemental analysis. The 1H
NMR spectrum of HP showed chemical shifts of the
phenylene group, benzothiadiazole group and alkylene
group at 8.1–7.7, 7.5–6.8, and 4.1–1.8 ppm, respectively.
The optical properties of the polymer were examined by
2.2. Synthesis of HP
2 (0.5 g, 0.925 mmol), benzene-1,4-diboronic acid
(0.233 g, 1.406 mmol), 3 (0.027 g, 0.0925 mmol), tris(4-
bromophenyl)amine (0.037 g, 0.154 mmol), an aqueous
2 M Na2CO3 solution (8 ml), and dry DMF (18 ml) under
nitrogen was added to a 100 ml round-bottomed flask.
After adding Pd(0) (5 mol%) as a catalyst the mixture was
ꢀ
heated to 90 C. The mixture was stirred for 40 h. The
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a UV-vis and fluorescence spectroscopy in 6 mM sodium
reaction mixture was poured into acetone. The precipitate
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phosphate buffer solution at a physiological pH of 7.4.
The polymer showed good solubility in the buffer solution
due to the presence of water-soluble sulfonic acid group
at the side chain of HP. Figure 1 shows the absorption
and photolumine-scence (PL) spectra of the hyperbranched
polymer in the solution state.
Copyright: American Scientific Publishers
was re-dissolved in deionized water and the solution was
dialyzed using a membrane (3500 cutoff) for 3 days. The
polymer was obtained after drying in freeze drier. Yield
1
0.21 g (40.2%). H NMR (300 MHz, D2O): ꢂ 8.1–6.8 (m,
8 H), 4.1–3.8 (m, 6 H), 3.3–2.8 (t, 6 H), 2.3–1.8 (d, 12 H)
ppm. FT-IR (cm−1ꢃ: 724 (S–O), 1151 (aryl C–N), 1372
(S O), 1618 (C C), 2939 (C–H), 3418 (OH).
The absorption maximum of HP in a buffer solution
was observed at 352 nm. This absorption corresponds to
the push-pull structure of HP, which has an electron-
donating triphenylamine group and an electron-accepting
benzothidaizole group. On the other hand, the maximum
emission of HP was observed at 418 nm. This was
3. RESULTS AND DISCUSSION
Scheme 1 illustrates the synthetic procedures for the prepa-
ration of the monomers and polymer. Monomers 1 and 2
for the preparation of the polymer were synthesized using
the literature procedures.23 Monomer 3 was obtained easily
in good yield by the bromination of commercially avail-
able 2,1,3-benzothiadiazole according to the published
methods.24 The hyperbranched polymer was typically pre-
pared by the palladium-catalyzed Suzuki cross-coupling
of diboronic acid, dibromo compound and tribromo com-
pound. For the preparation of HP, a slight excess of 1,4-
phenylenediboronic acid was used in the presence of an
additional catalytic amount of Pd(0) to produce a high
abundance of boronic acid end groups at the polymer
branches.
600
400
200
0
400
500
600
Wavelength (nm)
The reaction time for polymerization was determined
experimentally before the appearance of the precipitates.
HP obtained before precipitation had good solubility
Fig. 2. Fluorescence spectra of HP (1ꢄ0 × 10−5 M) upon the addition
of mannose (2ꢄ0 × 10−4 M) in 6 mM aqueous sodium phosphate buffer
at pH 7.4; ꢅex = 352 nm.
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Kim et al.
Fluorometric Detection of Lectin with Water-Soluble Hyperbranched Conjugated polymer Using Mannose Mediation
OH
Br
OH
O(CH2)4SO3H
Br
1,4-butanesultone
Br2
Br
Br
HO
HO
HO3S(H2C)4O
1
2
S
S
N
N
N
N
Br2
Br
Br
3
Br
Br
HO
B
HO
OH
OH
Pd(0)
N
2
+
3
+
B
+
aq.Na2CO3, THF
Br
OH
HO B
N
S
N
HO3S
OH
HO B
O
O
SO3H
N
S
N
SO3H
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S
N
N
O
Copyright: American Scientific Publishers
OH
B
N
HO3S
OH
O
O
O
SO3H
HO3S
N
N
SO3H
O
HO3S
O
O
SO3H
O
N
S
N
N
S
SO3H
N
B OH
HO
HO B
OH
HP
Scheme 1. Synthetic routes for the monomers and HP.
attributed to effective excitation energy transfer from the
short conjugated species to the larger ones, which are the
main emitting species.
Based on the well-known mannose-boronic acid
interaction, mannose-modified HP was prepared from a
reaction with boronic acid functionalized HP and mannose
at room temperature. The fluorescence intensity of HP
(1ꢄ0 × 10−5 M) decreased slightly upon the addition of
mannose, as shown in Figure 2. This suggests that the
introduction of mannose at the ends of HP did not affect
J. Nanosci. Nanotechnol. 12, 4365–4369, 2012
4367

Fluorometric Detection of Lectin with Water-Soluble Hyperbranched Conjugated polymer Using Mannose Mediation
Kim et al.
0.6
the ꢀ-conjugated structure because the fluorescence prop-
erty of the polymer had not been altered considerably.
Based on the complex formation between boronic acid and
diol groups in mannose, HP has a great advantage over its
0.4
linear counterpart, because a large number of end groups
(boronic acid) can be incorporated in the backbone.
Lectins have specific binding affinity to glucose and
0.2
mannose. Based on this binding property, an attempt
was made to add some proteins (5ꢄ0 × 10−7 M) in the
mannose-mediated HP buffer solution. Mannose-mediated
0.0
0.0
HP showed the most sensitive fluorescence intensity
0.5
1.0
1.5
2.0
2.5
3.0
change to lectin even in the ꢆM range (Fig. 3(a)).
Compared to lysozyme and cyt c (Figs. 3(b) and (c)),
a consecutive increase in fluorescence intensity was
log [protein]
Fig. 4. Emission intensity changes (I/I0ꢃ of mannose-mediated HP as
a function of the proteins concentration; 1ꢄ0×10−5 M in 6 mM aqueous
sodium phosphate buffer at pH 7.4; ꢅex = 352 nm and ꢅem = 418 nm for
HP; ꢀ lectin; ꢁ lysozyme; I0: fluorescence intensity before the addition
of the protein; ꢇI: fluorescence intensity change before and after the
addition of the protein.
(a)
800
600
400
200
0
observed in HP with increasing lectin concentration.
On the other hand, the change in fluorescence intensity
was negligible in the case of lysozyme due mainly to
the low binding affinity between lysozyme and mannose-
mediated HP. In the case of cyt c, the fluorescence was
quenched completely in the buffer solution as shown in
Figure 3(c). It was reported that the heme group in cyt c,
which is a complex compound with Fe2+ ions, can result in
400
500
600
Wavelength (nm)
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fluorescence quenching.25ꢁ26 This suggests that a number
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of boronic acid functional group on HP provide a potential
Copyright: American Scientific Publishers
(b)
600
400
200
0
binding with mannose, which can have a high selectivity
with lectin over other proteins as shown in Figure 4.
4. CONCLUSION
A blue-emitting hyperbranched water-soluble conjugated
poly(p-phenylene-benzothiadiazole) with a number of
boronic acid functional groups in its ends was synthe-
sized via Suzuki coupling polymerization. After bind-
ing to boronic acid end-groups in HP and mannose,
the polymer played a role in the selective sensing
of the mannose-binding glycoprotein, lectin. According
to the investigations on its sensing properties toward lectin,
the mannose-modified hyperbranched polymer can be used
as a selective and sensitive sensor for lectin.
400
500
600
Wavelength (nm)
(c)
600
400
200
0
Acknowledgment: Financial support from the National
Research Foundation (NRF) grant funded by the
Korea government (MEST) (2010-0022667) is greatly
acknowledged.
400
500
600
Wavelength (nm)
References and Notes
Fig. 3. Fluorescence spectra of mannose-mediated HP (1ꢄ0 × 10−5 M)
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Received: 13 May 2010. Accepted: 20 July 2011.
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4369