Dynamic glycovesicle systems for amplified QCM detection of carbohydrate-lectin multivalent biorecognition

Article abstract of DOI:10.1039/b924766a

We describe multivalent biorecognition of adsorbed lectin layers by biomimetic sensing nanoplatforms based on dynamic glycovesicles in a continuous flow QCM setup. The Royal Society of Chemistry.

Full text of DOI:10.1039/b924766a

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Dynamic glycovesicle systems for amplified QCM detection
of carbohydrate-lectin multivalent biorecognitionw
a
b
a
Eugene Mahon, Teodor Aastrup and Mihail Barboiu*
Received (in Cambridge, UK) 24th November 2009, Accepted 16th January 2010
First published as an Advance Article on the web 27th January 2010
DOI: 10.1039/b924766a
We describe multivalent biorecognition of adsorbed lectin layers
by biomimetic sensing nanoplatforms based on dynamic glyco-
vesicles in a continuous flow QCM setup.
determined. Thus QCM has been used in the areas of DNA
hybridisation, immunological systems and also in the areas of
1
4
protein–protein and protein–carbohydrate interactions.
A
general limitation, however, on sensor technologies sensitivity
is the mass of the analyte which is to be adsorbed from
solution. At present commercially available sensing systems
do not have intrinsic small molecule sensitivity. Amplification
methods may be employed in order to study certain biorecog-
nition processes. The most common amplification method is
Molecular recognition in biological systems occurs mainly in
interfacial environments such as at membrane surfaces, enzyme
active sites, or in the interior of the DNA double helix. At the
cell membrane surface, carbohydrate–protein recognition
principles apply to a range of specific non-covalent interac-
1
tions including immune response, cell proliferation, adhesion
2
and death, cell–cell interaction and communication. Protein–
3-dimensional surfaces, e.g. porous films, molecular imprinted
10c,17
polymers, multilayers etc.
A second strategy concerns the
3
protein recognition meanwhile accounts for signalling processes
4–7
and ion channel structure.
mass increase by the introduction of nanoplatforms such
16
15
as nanoparticles, vesicles to carry the recognising small
molecule element.
Surface based systems for biosensing in terms of real-time
label-free measurement of biointeractions have been extensively
developed which exploit electro-optical (Surface Plasmon
Resonance – SPR), piezoelectric (Quartz Crystal Microbalance –
QCM) and electrochemical transduction properties. The
piezoelectric phenomenon by which QCM sensors work is a
mechanical–electrical effect first reported in 1880, describing
the generation of electrical charges on the surface of solids
Vesicular aggregates exhibit an important advantage
as a biological sensing platform in that they mimic the cell
membrane – the site of molecular docking, ligand–receptor
binding and other vital natural processes such as exosomes. In
the last two decades an interest has developed in these
liposomal aggregates as drug delivery systems with their major
advantage being the physiological origin of their components
8
18
caused by pulling, pushing or torsion. It was later demon-
leading to high systemic tolerance. A third point is that they
strated by Sauerbrey that there exists a linear relationship
between mass adsorbed on piezoelectric crystal surfaces and
readily incorporate small molecular species either in their
aqueous cavity or lipophilic wall. Surveying specifically protein–
carbohydrate interaction studies the approaches used have been
9
the crystal’s resonant frequency in air or vacuum. Develop-
16
ment of this observation toward the study of biological
interactions was realised with the design of solution based
systems and combination of these with microfluidics and
surface chemistry. Consequently, QCM has become a highly
relevant analytical technique due to its sensitive solution–
surface interface measurement capability and so is highly
to modify the phospholipid building blocks or to incorporate
19–24
glycolipids or cholesterol carbohydrates in the bilayer.
Vesicles have been used to demonstrate molecular recognition
on the QCM platform for biotin–streptavidin affinity driven
25,26
adsorption.
Referring specifically to lectin recognition by small unilamellar
vesicles there are some noteworthy advantages to using
this platform in QCM: (a) the phospholipid surface is anti-
adhesive towards lectin proteins and so non-specific binding
1
0
applicable in label free analysis of biological binding events.
It possesses a wide detection range which at the low end can
detect monolayer coverage by small molecules. Although the
relationship described by Sauerbrey is only valid for a thin,
uniform, rigidly attached mass in vacuum, new models to
explain the mass–frequency relationship in liquid environ-
ments and for non-rigid materials were developed in later
2
7
can be avoided; (b) glycolipids should easily partition into
2
bilayers; (c) a small unilamellar vesicle has an average molar
8
6
ꢁ1 29
mass of around 1.5 ꢀ 10 g mol
.
Herein we report vesicle biorecognition of lectin layers in a
continuous flow QCM setup. Simple alkyl glycosides 1–4
(Scheme 1) were used to confer the highly specific ‘‘glycocode’’
to the recognising vesicle nanoplatforms which will present to
the multivalent lectin receptor Concanavalin A – Con A. This
strategy allows QCM investigation of processes with bio-
logically relevant approximation of in vivo parameters, thereby
providing a means to attaining an enriched understanding of
biorecognition events.
1
1–13
theoretical works.
Besides direct measurement of adsorbed
mass, concentration dependent measurements of the frequency
shift together with the assumption of a linear relationship
between DF and Dm allow thermodynamic and kinetic para-
meters of binding events as well as active concentrations to be
a
Institut Europe´en des Membranes – ENSCM-UMII-CNRS 5635,
Place Euge`ne Bataillon, CC 047, F-34095 Montpellier Cedex 5,
France. E-mail: mihai.barboiu@iemm.univ-montp2.fr
Attana AB, Bjo¨rnna¨sva¨gen 21, S-11347 Stockholm, Sweden
b
For these reasons, alkyl glycosides 1–4 were prepared
to act as synthetic molecular analytes.w They were prepared
using ‘‘click chemistry’’ i.e. glycosyl azides–tetradecyne
w Electronic supplementary information (ESI) available: Synthetic
and characterization details for 1–4. See DOI: 10.1039/b924766a
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Scheme 1 Alkyl glycosides for vesicle functionalisation: b-glucoside
, a-mannoside 2, b-galactoside 3, and b-maltoside 4.
Fig. 2 QCM adsorption profiles for alkyl glycoside functionalised
vesicles at different molar ratio of alkyl glycosides 1–4 to phospholipids
1
(
1:20/mol: mol = 10 mM in glycoside).
Huisgen-cycloaddition to give a 1,4-di-substituted 1,2,3-triazole
3
0–32
unit
with their anomeric configurations determined by
3
4
NMR. The vesicle solutions were prepared following a known
described for aryl glycosides. In any case, based on the
literature it would not be expected to have a negative effect
on binding.
3
3,
protocol for SUV’s. z The functionalised vesicles V1–V4
were prepared by incubation of the vesicular solutions with
different amounts of alkyl glycoside 1–4 for 24 h at 4 1C.y
Small unilamellar vesicles V1–V4 (25–35 nm in diameter) were
tested for interaction with the Con A layers by QCM.
Another interesting characteristic is that the glycosides 1–4
might present an enhanced fluidity in the phospholipids bilayer,
2
4
with reference to glycolipids or cholesteryl-carbohydrates.
With Con A layers prepared in situ on a QCM gold
electrode by specific recognition of a polymannoside surface
The ‘‘maltovesicles’’ V4 may allow a carbohydrate external
microdomain formation with multivalent presentation.
The result of fitting ‘‘maltovesicles’’ V4 adsorption on
Con A to the Langmuir model gives an association constant
(
see ESIw), vesicle solutions displaying an alkyl glycoside type
at different concentrations were injected into the QCM setup
Fig. 1). The vesicle solutions had a concentration of 200 mM
4
ꢁ1
34
(
of K
a
= 8.4 ꢀ 10 M (see ESIw). Con A is well known to
in POPC and various molar ratios of alkyl glycosides 1–4 to
selectively bind a-mannosides and a-glucosides as well as
3
5
phospholipid were tested.
substituted a-mannoside analogues. This demonstrated an
affinity enhancement when compared with the Me-a-Glc
monosaccharide which has shown in calorimetry experiments
As shown in Fig. 2 only ‘‘maltovesicles’’ V4 adsorbed
specifically while all the others V1–V3 showed little adsorption
up to the 10 mM concentrations of 1–3. The first point
of interest in these results is that the ‘‘mannovesicles’’ V2
3
ꢁ1
a K
a
= 2.4 ꢀ 10 M , even more so when compared with
3
ꢁ1 35a
.
D-maltose K
a
= 1.6 ꢀ 10 M
Me-a-Man shows a higher
1
0,24
3
ꢁ1
(
mannose: well known binder to Con A)
derived systems
affinity at 7.6 ꢀ 10 M while the corresponding b-glycosides
35b
of both Man and Glc showed no measurable affinity. This
40-fold increase can be attributed to multivalent glycoside
show no apparent affinity whereas the ‘‘maltovesicles’’ V4 do.
A likely cause for this is the accessibility or the orientation of
the alkyl glycosides 1–4 at the bilayer surface. The maltoside
ligand 4, bearing a terminal a-Glc moiety could protrude more
from the bilayer compared to the monosaccharide ligands 1–3,
which may be buried in the external (or internal) hydrophilic
part of the phospholipid layer. The triazole unit just next to
the glycoside may have an influence. It could be expected that
the triazole ring would confer stabilizing interactions between
aromatic aglycon and protein amino acids nearby as has been
3
6
presentation, allied to their fluidity within bilayers.
The dynamic hydrophobic interface between carbohydrate
molecules and bilayers might mediate the structural self-
correlation of supramolecular sugar clusters by virtue of their
basic constitutional behaviours. The resultant dynamic system
can undergo continual change in its constitution, through
dissociation/reconstitution of different mesophases during
3
7–39
the vesicle recognition process.
Their dynamic constitution
might render the emergence of recognition events; synergetic
and vesicular systems may adapt their external surface dis-
tribution allowing ConA to access more than one binding site
simultaneously. This concept can be related to a sort of
4
0a
‘
‘Chemical collectivism’’
0b–d
or to the ‘‘Dynamic interactive
4
systems’’
characterized by their aptitude to macroscopi-
cally organize (self-control) their distribution in response to
external stimuli in coupled equilibria.
In conclusion, signal amplification through coupling of
vesicular nanoplatforms to QCM has been demonstrated to
be a versatile method, especially given their facile preparation
and propensity to partition amphiphilic molecules. At present
commercially available QCM systems do not demonstrate
small molecule sensitivity and vesicles offer a route to large
Fig. 1 Con A layers exposed to functionalised vesicle solutions within
a QCM system.
2
442 | Chem. Commun., 2010, 46, 2441–2443
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mass amplification allied to their presentation of a dynamic
biomimetic interface. This work showed ligand design is, as
expected, an important feature and multivalent ‘‘biomimetic’’
longer chain glycolipids could show even higher affinity
amplifications.
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Chem. Commun., 2010, 46, 2441–2443 | 2443