Tunable, dynamic and electrically stimulated lectin-carbohydrate recognition on a glycan-grafted conjugated polymer

Article abstract of DOI:10.1039/c2cc31789c

An electroactive platform for multivalent, reversible and electrically stimulated lectin-carbohydrate recognition based on mannose-functionalized conjugated polymer is reported and tuned by electrocopolymerization of mixture of tri(ethylene glycol)-functi

Full text of DOI:10.1039/c2cc31789c

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COMMUNICATION
Tunable, dynamic and electrically stimulated lectin–carbohydrate
recognition on a glycan-grafted conjugated polymerw
Shyh-Chyang Luo,a Eric Assen B. Kantchev,z*b Bo Zhu,a Yuen Wee Siangb and
Hsiao-hua Yu*a
Received 10th March 2012, Accepted 18th May 2012
DOI: 10.1039/c2cc31789c
An electroactive platform for multivalent, reversible and electrically
stimulated lectin–carbohydrate recognition based on mannose-
functionalized conjugated polymer is reported and tuned by electro-
copolymerization of mixture of tri(ethylene glycol)-functionalized
EDOT and its a-mannose conjugate.
with electrical stimulation would be important to decode the
cellular–cellular communication within such supplications.
With the ultimate aim of developing such a platform, herein
we describe the specific, tunable, dynamic and electrically
stimulated recognition of a biomolecule (exemplified by a
plant lectin, Concanavalin A) and a conjugated polymer,
poly-(3,4-ethylenedioxythiophene) (PEDOT), the surface of
which is modified by the complementary ligand, a-mannoside.
PEDOT19 is a widely used organic electronics material and it
also demonstrates a great potential for biological applications
because of its superior biocompatibility and long-term stability
of its materials characteristics in aqueous solutions.20,21 So far,
only a few glycan-conjugated conjugated system have been
reported for biosensing application.22,23
Multivalent and oligomeric carbohydrates (glycans) found in
cell surface glycoproteins and glycolipids play an important
role in numerous normal and pathological biological events.1–3
Lectins are multimeric glycan-binding proteins that show a
significant increase of binding affinity per carbohydrate unit
with increase of glycan size (cluster glycoside effect).4 The
glycan–lectin recognition regulates such biological processes as cell
proliferation, adhesion, immune responses, cell–cell interaction
and communication, among others.5–8 Various glycan arrays
and glyconanostructures utilizing fluorescence,9,10 surface plasmon
resonance (SPR)11–13 and quartz crystal microbalance (QCM),14,15
among others, have been developed to measure the lectin–glycan
binding affinity, analyze recognition mechanism13 and perform
pathogen sensing.14,16
Starting from hydroxymethyl-functionalized 3,4-ethylene-
dioxythiophene (EDOT-OH, Chart 1), triethylene glycol
substitute monomer (EDOT-EG3) was synthesized (ESIw).
This moiety was chosen because oligoethylene glycol could
increase the hydrophilicity of the surface and prevent
unwanted non-specific binding of proteins and cells.24,25 After the
compound was synthesized, a-mannoside can be easily conjugated
to yield the desired monomer, EDOT-Man. Conjugated polymers
poly(EDOT-Man) obtained from electropolymerization of EDOT-
Man showed considerably stronger binding to Concanavalin
A (Con A) than poly(EDOT-OH) and Au substrates (ESIw).
Moreover, poly(EDOT-EG3) displayed reduced non-specific
binding of bovine serum albumin (BSA) compared to
poly(EDOT-OH) because of the greater hydrophilicity.
The surface functional group density for functionalized
PEDOTs prepared from microemulsion electropolymerization
Electrically active platforms based on conjugated polymers
have been utilized to understand and evaluate biomolecular
recognition events, including complementary DNA binding
and aptamer/protein recognition due to the great current
interest for the development of low cost, portable lab-on-a-chip
bioanalytical and diagnostic devices. The connection between
glycans and pathological events allows this pair to be a promising
target. In addition, there has been a growing interest to study
the electrical stimuli on physiological characteristics of cells, such
as neurons, for potential application in tissue engineering.17,18
Integration of the glycan-mediated cellular information
is usually within the range of 1015 cmꢀ2 26
At this relatively
.
high surface density of mannose functional groups, multivalent
carbohydrate–lectin recognition with a high apparent binding
constant is expected.11,13,15 This inhomogeneous binding event
a Yu Initiative Research Unit, RIKEN Advanced Science Institute,
2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
E-mail: bruceyu@riken.jp; Fax: +81(0)48-462-1659;
Tel: +81(0)48-467-9513
b Institute of Bioengineering and Nanotechnology, 138669, Singapore
w Electronic supplementary information (ESI) available: Synthetic
procedures and characterization data for the monomers, electropoly-
merization curve and surface morphology by AFM and QCM data.
See DOI: 10.1039/c2cc31789c
z Current address: Institute of Materials Research and Engineering,
3 Research Link Singapore 117602. Fax: +65 6872 0785;
Tel: +65 6874 1324. E-mail: kantcheveab@imre.a-star.edu.sg.
Chart 1
c
6942 Chem. Commun., 2012, 48, 6942–6944
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may lead to complex and unpredictable surface properties that
are inferior for diagnostic or bioanalytical devices.
biointerface was achieved by ‘‘washing off’’ with methyl-a-
D-mannoside solution. The release of Con A from the surface was
mainly due to the competitive inhibition binding. However, the
real-time monitoring by the quartz crystal microbalance (QCM)
indicated that a certain number of biomolecules were still bound
to the surface (step (v) in Fig. 2(b)). The residual biomolecules
could be washed off upon further rinsing with Tris buffer, hence it
was suspected that these molecules were methyl-a-D-mannoside
physically adsorbed on or hydrogen-bonded to the surface.
Adsorption of methyl-a-D-mannoside was also detected on freshly
prepared poly(EDOT-EG3)-co-(EDOT-Man) surfaces (Fig. 2c).
The binding and releasing event can be repeated consistently,
indicating a well-controlled surface for lectin–carbohydrate
recognition.
For example, treatment of poly(EDOT-Man) with
a
competitive inhibitor, methyl-a-D-mannoside (100 mM) in a
quartz crystal microbalance (QCM) chamber showed a strong
retention of Con A on the chip surface. The mannosylated
PEDOT surface was irreversibly blocked by Con A due to the
strong multivalent recognition. In that case, surface-bioconjugated
poly(EDOT-Man) was limited to a single use in biosensing and
other bioengineering applications. From a practical point of view,
a recyclable and reusable device is highly desirable. To that end,
the apparent binding affinity needs to be optimized to a range
where it is sufficiently strong to induce a signal, yet sufficiently
weak to ensure reversibility of the binding. On this platform, this
goal can be achieved by electropolymerization of a mixture of
conjugated and non-conjugated monomers to adjust the surface
density of the mannose functional group, thereby optimizing the
surface affinity for Con A.
The advantageous feature of a recognition event on a
conjugated polymer is that it provides an electroactive platform
to study both the recognition event with an electrical signal and
the electrical stimulation effect on the biological interface.
When a potential of ꢀ200 mV (vs. Ag/AgCl) was applied to
the substrates, the quantity of Con A bound to the surface
almost doubled compared to the substrate without applying any
electrical stimulation, as shown in Fig. 3a.
Non-specific binding of bovine serum albumin (BSA) and
specific binding of Con A were evaluated for two copolymers,
poly(EDOT-OH)-co-(EDOT-Man) and poly(EDOT-EG3)-co-
(EDOT-Man) with EDOT-Man molar composition in monomer
solution mixture ranging 0–100%, as shown in Fig. 1. In general,
the binding of Con A increased with a higher EDOT-Man
composition percentage. At the same time, the non-specific binding
of BSA was greatly reduced with increasing EDOT-Man monomer
percent in poly(EDOT-OH)-co-(EDOT-Man). This was mainly
due to the greater hydrophilicity provided by mannose functional
groups to prevent non-specific binding. On the other hand,
non-specific binding of BSA with poly(EDOT-EG3)-co-(EDOT-
Man) was further reduced compared to poly(EDOT-OH)-co-
(EDOT-Man). As shown in Fig. 1b, the binding of Con A on
poly(EDOT-EG3)-co-(EDOT-Man) experienced a dramatic and
non-linear increase up to about 50% EDOT-Man monomer
content and levelled off thereafter. It was also intriguing to observe
that the Con A binding was greater on poly(EDOT-EG3)-co-
(EDOT-Man) compared to it on poly(EDOT-OH)-co-(EDOT-
Man). This indicated that the specific binding was enhanced by the
more hydrophilic surface.
Evaluation of the electrical stimulation during the Con A
binding in situ by applying alternating potentials of ꢀ200 mV and
0 V for 20 s, respectively, clearly displayed that the frequency
dropped immediately after the electrical pulse was applied. This
indicated more adsorption of Con A (Fig. 3b and c). Con A
binding when a positive potential from 0 to 500 mV (vs. Ag/AgCl)
was applied did not show a difference from the non-electrically-
stimulated sample. A control experiment showed only little
frequency decrease when an identical electrical pulse was applied
Poly(EDOT-EG3)-co-(EDOT-Man) prepared at the optimal
50% EDOT-Man monomer content proved to be capable of
repeated Con A recognition (Fig. 2a). Recovery of the active
Fig. 2 Real-time monitoring of repeatable Con A recognition by
QCM: (a) and (b) cycles of Con A binding and releasing on
poly(EDOT-EG3)-co-(EDOT-Man); (c) adsorption of methyl-a-
D-mannoside on poly(EDOT-EG3)-co-(EDOT-Man). The real-time
experiment steps are as following: (i) addition of BSA (1 mg mLꢀ1),
(ii) rinsing with Tris-buffer, (iii) binding of Con A (1 mg mLꢀ1),
(iv) rinsing with Tris-buffer, (v) releasing Con A in the presence of
methyl-a-D-mannoside (100 mM) and (vi) rinsing with Tris-buffer.
Fig. 1 Protein binding of non-specifically targeted BSA (’) and
specifically targeted Con A ( ) on (a) poly(EDOT-OH)-co-(EDOT-
Man) and (b) poly(EDOT-EG3)-co-(EDOT-Man). The mass of the
protein binding layer was estimated by Sauerbrey relationship27 based
on 3rd overtone data obtained from the quartz crystal microbalance
(QCM).
c
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controlled by using electrical stimulation. By applying a
potential of ꢀ200 mV (vs. Ag/AgCl), the binding of Con A
was enhanced dramatically. Importantly, Con A can be
removed by methyl-a-D-mannoside solution, allowing for the
substrate to be recycled for multiple binding events.
This work was funded by RIKEN Advanced Science
Institute and Grant-in-Aid for Young Scientist (Nos. 22681016
and 23710138) from JSPS/MEXT. This work was also supported
in part by Institute of Bioengineering and Nanotechnology
(Biomedical Research Council, Agency for Science, Technology
and Research, Singapore).
Notes and references
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In summary, we have prepared an electrically active platform
with glycan-grafted conjugated polymers. The surface density of
the mannose ligand that is utilized for this mimic can be easily
adjusted by controlling the composition of the mannosylated
monomer, EDOT-Man. The lectin–carbohydrate interaction is
affected by the surface composition of both the ligand density
and surface hydrophilicity. The binding event can be further
c
6944 Chem. Commun., 2012, 48, 6942–6944
This journal is The Royal Society of Chemistry 2012