Int. J. Nanotechnology, Vol. x, No. x, xxxx
1
Biomimetic membranes and biomolecule
immobilisation strategies for nanobiotechnology
applications
Agnes P. Girard-Egrot, Christophe A.
Marquette and Loïc J. Blum *
Laboratoire de Génie Enzymatique et Biomoléculaire
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires
ICBMS; UMR5246; Université Lyon1 – CNRS
43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France
Fax: +334 72 44 79 70
E-mail: [email protected]; [email protected]; [email protected]
*Corresponding author
Abstract: Biological membranes constitute a source of inspiration for making
supramolecular assemblies which can be used in the design of biomimetic
sensors. At the same time, the concept of using biomolecules as an elementary
structure to develop self-assembled entities has received considerable attention.
More particularly, the ability of amphiphilic molecules like lipids, to
spontaneously organize into bilayers, is suitable to achieve biomimetic
membrane models.
The potential of two-dimensional molecular self-assemblies is clearly illustrated
by Langmuir monolayers of lipids formed at an air/water interface, which can
be used as models to acquire knowledge about the molecular recognition
process occurring in biological membranes. Langmuir-Blodgett (LB)
technology, based on the transfer of this interfacial monomolecular film onto a
solid support, allows building up lamellar lipid stack, with an accurate control of
thickness and molecular organisation. This technique offers the possibility to
prepare ultrathin layers suitable for biomolecule immobilization.
We are presenting herein an overview of work performed in our group that
sheds light on the formation of biomimetic LB membranes associating protein
in a well-defined orientation. Two points will be addressed: investigations of
protein/lipid interactions using lipid monolayers as membrane models and
biosensing applications. The objectives are to highlight advantages of interfacial
Langmuir monolayers and supported Langmuir-Blodgett films to investigate
molecular interactions between biomolecules and lipid membrane components
or to elaborate biomimetic membranes as sensing layers, respectively.
The present article also draws a general picture of non-conventional methods for
biomolecule immobilization and their applications for biochip developments.
The technologies presented are based either on original solid supports or on
innovative immobilization processes. First, “Macromolecules to PDMS
transfer” technique relying on the direct entrapment of macromolecules spots
during PDMS polymerisation is proposed as an alternative for the easy and
simple PDMS surface modification. Then, the electro-addressing of
biomolecule-aryl diazonium adducts at the surface of conducting biochips will
be presented and shown to be an interesting alternative to immobilization
Copyright © 200x Inderscience Enterprises Ltd.
2
processes based on surface functionalization
Keywords: Biochip; Biomimetic membrane; Microarray; Langmuir-Blodgett
film; Lipid bilayer;
Reference for publisher use only
Biographical notes:
Prof. A.P. Girard-Egrot received the Doctorat de spécialité in Biochemistry
(1995) and the “Habilitation à Diriger des Recherches” (2002) from Université
Claude Bernard-Lyon 1. She is presently Professor of Biochemistry at the same
university and is in charge of development of biomimetic membranes based on
Langmuir-Blodgett technology. Since 1993 author or co-author of more than 25
articles and book chapters.
Dr. C.A. Marquette received the Doctorat de spécialité in Biochemistry (1999)
from the Université Claude Bernard-Lyon 1. He is presently permanent
researcher at the Centre National de la Recherche Scientifique (CNRS) at the
UCBL and is in charge of the development of optical biochips and micro-arrays
based on luminescent reactions. Since 1998 author or co-author of more than 50
articles and book chapters.
Prof. L.J. Blum received the Doctorat d’Etat ès Sciences (1991) from the
Université Claude Bernard-Lyon 1. He is presently Professor of Biochemistry
and Biotechnology at the same University and is involved in the development of
biosensors, bioanalytical micro and nano systems and biomimetic membranes.
He is the head of the UCBL/ CNRS research unit ICBMS. Since 1983 author or
co-author of 150 articles and book chapters.
1
Biomimetic membranes for nanobiotechnology applications
Biological membranes play a central role in the cell life. Besides their compartmenttalization function, they are involved in many exchange processes between the outside
and inside cellular worlds.
Only a few manometers thick, biological membranes consisted of two main components,
have a perfect organization on the molecular level. Lipids, held together by hydrophobic
interactions, essentially play the structural role, forming a continuous bilayer acting as a
diffusion barrier. Proteins, like transmembrane proteins or peripheral membrane proteins,
respectively embedded within the membrane or transiently associated with it, are devoted
to either exchange or biocatalysis processes. Consequently, biological membranes, highly
complex and dynamic supramolecular structures, are a key component of the way that
living cells are able to maintain and organize their function. Unlocking the secrets of
those membranes provides important lessons that are valuable in guiding the construction
of devices to be used for nanotechnological applications [1]. In particular, molecular and
supramolecular understanding of the architecture of biological membranes constitutes an
extraordinary source of inspiration for making ‘intelligent’ nanostructures which can be
used in the design of biomimetic sensors based on the molecular recognition and signal
transduction events occurring in natural membranes. Nowadays, direct contact between
nanostructures mimicking cell membranes and electronic devices offers a direct way of
3
following molecular processes (or biocatalytic reactions) by studying a very limited
number of molecules. Actually, bioelectronic interfacing between living and inert matter
lies at the heart of nanobiosciences. In this sense, biomimetic membranes provide basic
support structure for many applications in nanobiotechnology.
At the same time, access to the complex functioning of biological membranes is a real
challenge. Exploratory studies are performed both on integrated and on reconstituted
systems using models of natural membranes. Hence, since the complexity of cell
membranes, reliable models to acquire current knowledge of the molecular processes
occurring at biological membranes, either for studying basic membrane processes or for
technological applications need to be achieved.
Natural membrane organization builds upon the self-association properties of biological
molecules making them up. In vitro, these provide the basis for a natural and spontaneous
formation of bilayer structures which can be exploited to reconstitute biomimetic
membranes and a wide range of protein-lipid nanostructures.
Hence, the concept of using biomolecules as an elementary structure to develop selfassembled entities corresponding to organized supramolecular arrangements has thus
received considerable attention [2]. More particularly, the self-assembly ability of
amphiphilic biomolecules such as lipids, to spontaneously organize into nanostructures
mimicking living cell membranes, has appeared as a suitable concept for the development
of biomimetic membrane models [3-5]. At the same time, the growing interest in
confining lipid membranes on solid support has been nourished by the emergence of a
wide range of surface-sensitive characterization techniques which can be applied to study
model characteristics or proteins/membrane interactions.
The potential of two-dimensional molecular self-assemblies is clearly illustrated by
Langmuir monolayers of lipid molecules formed at an air/water interface, which can be
used as models to understand the role and the organization of biological membranes [6],
or to acquire knowledge about the molecular recognition process [7-9]. LangmuirBlodgett (LB) technology, based on the transfer of this interfacial monomolecular film
onto a solid support, allows building up lamellar lipid stacks. When all transfer
parameters are optimized, this technique corresponds to one of the most promising for
preparing supported lipid membranes as it enables (i) an accurate control of the thickness
and of the molecular organization, (ii) an homogeneous deposition of the monolayer over
large areas compared to the dimension of the molecules, (iii) the possibility to transfer
monolayers on almost any kind of solid substrate and (iv) to elaborate bilayer structures
with varying layer compositions.
Based on the self-assembled properties of amphiphile biomolecules at the air/water
interface, LB technology offers the possibility to prepare ultrathin layers suitable for
immobilization of bioactive molecules.
Here we present an overview of work performed in our group that sheds light on the
formation of biomimetic LB membranes associating protein in a well-defined orientation.
Two points will be addressed: biosensing applications and investigations of protein/lipid
interactions using lipid monolayers as membrane models. The objectives are to highlight
advantages of interfacial Langmuir monolayers and supported Langmuir-Blodgett films
to investigate molecular interactions between biomolecules and lipid membrane
components or to elaborate biomimetic membranes as sensing layers, respectively.
4
1.1 Langmuir monolayer formation and Langmuir-Blodgett technology
Langmuir monolayer technique is based on the properties of amphiphile biomolecules
like lipids to orient themselves at an air/water interface and to form an insoluble
monolayer called Langmuir film (Figure 1). When amphiphile molecules are deposited on
the surface of water, the dispersion forces quickly cause the solution to spread over the
whole available surface. After solvent evaporation, lipid headgroups are immersed in the
subphase, the hydrophobic tails pointing toward the gaseous phase. The interfacial film
resulting is a monomolecular layer of one-molecule thick.
The monolayer, initially present in a gaseous phase, is then compressed by two
mobile barriers. As the available surface area of the monolayer is reduced, the molecules
start to interact and the surface tension lowered. During compression, the molecules selfassembled at the interface to form a homogeneous interfacial film. The condensation state
of the molecules is directly correlated to the surface pressure (π) increase recorded by a
Wilhelmy plate partially immersed through the interface. The surface pressure (π)
corresponding to the force exerted by the film per unit length equals:
π = γ0 - γ
(1)
where γ0 is the surface tension of the pure liquid and γ the surface tension in the presence
of a monolayer.
During this process, the hydrophilic and hydrophobic ends of the molecule ensure that the
individual molecules are aligned in the same way.
Figure 1
Langmuir monolayer
formation
Surface pressure (π)
measurement
1. Molecules spread at the air/water interface
Wilhelmy plate
(tensiometer)
Formation of a monomolecular film in a gaseous state
Õ
LB trough
2. Monolayer compression by two mobile barriers
πÒ
Molecular self-organisation at the interface
and formation of an interfacial film in
different aggregation states
Õ
3. Monolayer in condensed state
Monomolecular film perfectly organised at the
air-water interface at the end of compression
Õ
When the surface pressure is sufficiently high to ensure lateral cohesion in the interfacial
film, the floating monolayer can be transferred, like a carpet, from the water surface onto
a solid support (Figure 2). During the transfer, the surface pressure is maintained constant
by a feedback servoloop of the compression system.
In the Langmuir-Blodgett technology [10, 11], the film deposition arises from the vertical
movement of a solid substrate through the monolayer/air interface. Depending on
5
whether the substrate is hydrophilic or hydrophobic, the first monolayer will be
transferred as the substrate is respectively raised or lowered through the interfacial film.
Subsequently, bi- or multi-layer stacks, called Langmuir-Blodgett films, are produced by
deposition of one monolayer each time the substrate goes through the interface. Hence,
the lamellar arrangement is representative of the natural lamellar stack of the biological
membrane.
The LB technique offers the possibility to perfectly control each step of LB films
formation. The main advantage with LB membranes lies in the highly ordered molecular
arrangement, achieved on the water surface and conserved during transfer onto the
substrate when all transfer parameters (surface pressure, rate of immersion of the
substrate, temperature, composition of the aqueous phase) have been optimized [12-14].
Akin to the biological bilayer, the structure of LB films makes them candidate for
developing biomimetic models of natural membranes.
Figure 2
Langmuir-Blodgett deposition
Vertical transfer onto a solid support
Constant surface pressure
Hydrophobic support
Hydrophilic support
Lamellar arrangement
1.2 Investigations of molecular interactions in Langmuir monolayers as
membrane model
As previously shown, Langmuir monolayers are monomolecular films formed at the
air/water interface. Due to their organization similar to the leaflet of the natural
phopholipidic bilayer, phospholipid monolayers represent in turn an attractive membrane
model, the thermodynamic relationship between monolayer and bilayer membranes being
direct [15].
6
Figure 3
Langmuir monolayer as membrane model
Surface pressure (π)
measurement
Ù
ª
ª
Biological membrane leaflet
Biomimetic membrane model
The main advantages of this model are the achievement of a molecular state perfectly
organized at the water surface and the control of the aggregation state by the lateral
pressure imposed to the lipids in well-defined physicochemical conditions (subphase
composition, pH, ionic strength, temperature) [16]. Mainly, monolayers overcome the
limitation to regulate lipid lateral-packing density and lipid composition independently,
as encountered for liposome dispersions. Especially, all the molecules forming the
monolayer are in the same orientation (absence of curvature or constraints at the level of
the phospholipid polar groups). Hence, Langmuir monolayers have been extensively used
for studying membrane interactions of peptides [17-19] or proteins [9]. Generally
speaking, Langmuir monolayers may be used to characterize the penetration capacity of
biomolecules in a lipid membrane at a molecular scale. They are also informative on the
stability of the molecule in the lipid environment.
Figure 4
Principles of molecular interaction investigations between biomolecules and Langmuir
monolayer
Molecule injection under monolayer at
constant surface area
Surface
Pressure (π)
πÒ
∆π
π initial
Ö
Injection
Time
ª
Surface
Pressure (π)
Ö
Analysis of penetration kinetics
∆π
(∂π/∂t)t=0
Initial velocity of surface
pressure increase
Extrapolation of the linear
plot at ∆π = 0
π initial
Time
ª
Surface pressure (π) N
Affinity of the protein for the monolayer
ª
Exclusion surface pressure :
“penetration capacity”
7
Langmuir monolayer allows the investigation of molecular interactions between
biomolecules and membrane components forming the membrane leaflet when a molecule
is injected in the aqueous phase under the monolayer compressed at a defined surface
pressure (π initial). If it inserts itself into the interfacial film, thus indicating an interaction,
the surface pressure increases, provided that the area is held constant (Figure 4). This
topography allows simulating, under realistic biological conditions, what happens when
hydrosoluble molecules (peptides, cytoplasmic proteins, hormones, probes, etc.) interact
at the surface of target cell (or organelle) membranes.
The molecular interactions with the interfacial film lead to a time-dependent surface
pressure increase (when the monolayer surface area is fixed). The analysis of the
penetration kinetics allows getting insights in both the penetration capacity and the
affinity of the molecule for the lipid constituting the monolayer. Moreover, the variation
of the surface pressure in the interfacial film after insertion is an indicator of the stability
of molecules in the lipid environment.
At the same time, the surface morphology of the film inserting the molecule may be
observed by Brewster Angle Microscopy (BAM) [20-23]. BAM is a powerful in situ
surface investigation technique which allows a direct visualization of the lipid domain
morphology formed in the monolayer at the air/water interface. It gives some information
about the homogeneity of the interfacial film. This technique is informative on the
structural rearrangement which can occur during interaction of molecules with lipid
monolayer. In this context, BAM may be useful to know if the interacting molecule can
modify or not lipid membrane organization.
8
Figure 5
Morphology of mixed PC:PE (2:1) monolayers in interaction with CRMP5.
BAM image shows the morphology of the proteo-lipidic monolayer at the air /buffer
interface. AFM images were taken after transfer of the monolayer onto a silica substrate
at a surface pressure of 21 mN/m. Height differences confirms exclusion of CRMP5
from the condensed domains and shows that it mainly concentrates at the periphery of
condensed domains.
BAM image
21 mN/m
AFM images
Condensed lipid domains
of ≈3 nm height
2 µm
5 µm
50 μm
10 µm
Mixed proteo-lipid domains
of ≈6 nm height
350 µm
For instance, we have recently investigated in our group, the interaction of CRMP5 [24],
a member of collapsing response mediator protein (CRMP) family, with phospholipid
Langmuir monolayer as model to estimate its insertion ability. The collapsin response
mediator proteins are strongly expressed in the developing brain where they take part in
several aspects of neuronal differentiation. Their expression is down-regulated in the
adult brain, but, CRMPs are expressed again with high levels during some cancers or
neurodegenerative diseases. Among them, the role and the biochemical functions of the
most recently discovered CRMP5 remain obscure. While present in cytosol, CRMP5 has
been also localized in some cell membrane fractions. Using Langmuir monolayers, we
have showed that CRMP5 is able to penetrate into both monolayers composed of PC1 or
PE2, used as phospholipids representative of the biological membranes (data not shown).
With a mixed monolayer composed of PC and PE at a 2:1 molecular ratio, BAM and
AFM techniques demonstrate that CRMP5 localizes preferentially in the fluid phase of
the monolayer with a high tendency to exclude from the condensed phase (Figure 5). This
study confirms CRMP5 potentiality for interacting with cell membranes and illustrates
1
2
Phosphatidylcholine
Phosphatidylethanolamine
9
the potentiality of the Langmuir monolayer as membrane model to investigate
lipid/protein interactions or macromolecules in general.
1.3 Functionalised Langmuir-Blodgett lipid films as supported biomimetic
membrane models: applications in Nanobiotechnology
Lipid membranes are self-assembled entities that can be used in a general manner as
substrates for the immobilization of biomolecules which may have specific biological
activities. The functionalisation of lipid membranes to develop protein–lipid assemblies
is a crucial step in many applications in nanobiotechnology.
The functionalisation of Langmuir-Blodgett lipid films can be achieved by association of
proteins presenting specific recognition properties, such as enzymes, antibodies, receptors
or specific ligands, in order to develop ordered protein–lipid molecular assemblies. These
supported biomimetic membranes, corresponding to supramolecular arrangements, can be
used to functionalise surfaces, upon which the protein confers its biospecificity.
Over the past twenty years, a lot of research has been carried out on the association of
proteins, and in particular enzymes, with Langmuir–Blodgett films. The bioactive films
obtained in this way have been studied for their potential applications in the design of
biosensors, with the protein–lipid LB membranes integrated into these systems as
ultrathin sensitive films [25]. Since they can be transferred on various types of substrate,
these films exhibit many advantages for the development of novel micro- or
nanobiosensors, inspired by biological models. Especially, biomimetic biosensors based
on the direct transduction of biological signals like in the biological membrane can be
produced. As for other systems mimicking biological membranes, their structural
organisation (highly ordered) and their ultrathin dimensions (a few nanometres thick) are
the main characteristics for designing micronic sensors operating on the molecular scale
and displaying ultra-rapid response times, fundamental criteria for further development of
‘smart’ sensors or biochips. However, the interest of LB films is not limited to these
structural aspects. Specific advantages are worth mentioning: (i) the elaboration of a
bioactive sensing layer and its association with the transducer is achieved in a one-step
procedure, (ii) only a very small amount of protein is required to prepare the membrane,
(iii) experiments are performed at ambient pressure and temperature hence avoiding the
kind of thermal treatments required in the design of electronic systems, which would
damage biological components, (iv) the performance of the sensor in terms of detection
limit, sensitivity and dynamic range can be modulated by varying the number of the
deposited protein–lipid layers [26-29].
The crucial stage in the fabrication of supported biomimetic LB membranes remains the
incorporation of the biological element in LB films, without alteration or loss of activity.
Several methods have so far been developed to produce active self-associated protein–
lipid assemblies in bilayers or multilayers [25]. The most commonly used derives from
the procedure developed to study protein–lipid interactions with a Langmuir monolayer
[15, 30, 31]. It corresponds to the adsorption of the protein present in the subphase onto
the interfacial film before transfer of the mixed proteo-lipidic monolayer [27, 30, 32-43].
This method is particularly well suited to extrinsic peripheral proteins capable of
associating with biological membranes or to anchoring proteins inserting themselves into
one leaflet of a bilayer. However, it presents some drawbacks [44]. The presence of the
10
protein in the interfacial film may affect its transferability properties [26, 30, 45]. The
surface pressure required for the transfer procedure may not be always well suited for the
enzyme association (ejection of the protein at high surface pressure). The presence of the
protein may induce a poor monolayer adhesion on the substrate, leading to a peeling-off
at the subsequent immersion.
Another approach consists in adsorbing the protein onto pre-formed LB film [46-50]. The
main advantage with this procedure lies on the possibility of associating the protein with
a hydrophilic lipid surface (polar head at the surface) or a hydrophobic lipid surface
(hydrocarbon chains at the surface), depending on the number of layers deposited on the
substrate. Nevertheless, the interactions involved in this type of association are often too
weak to prevent the release of protein molecules which remains a major drawback as
previously shown in our group [51] or reported elsewhere [52], and is often the main
reason explaining the poor reproducibility of responses of LB membrane-based sensors.
In order to minimise desorption of protein molecules, some authors have suggested to
covalently immobilise the protein on LB film surfaces by means of cross-linking agents
[53, 54]. The stabilisation of the proteo-lipidic LB films by reticulation after transfer with
glutaraldehyde vapour has been also investigated [45, 55, 56]. The fact remains, however,
that covalent attachment to the lipid structure may induce changes in the protein
conformation, which may cause a loss of its biological activity.
Another alternative for limiting desorption and avoiding covalent immobilisation of the
protein has been proposed in our group. It consists in covering the protein molecules by
transferring a further lipid layer onto the surface of the adsorbed molecules [46, 57-59].
This procedure referred to as “inclusion process” allows the sandwiching of the enzyme
in a hydrophobic or a hydrophilic environment while keeping the homogeneity of the
supporting layers. The specific features of these latter methods are the possibility to
easily modify the lipid composition of the protective leaflet [60] and, to some extent, to
reproduce the membrane asymmetry which can favour the physical retention of the
protein and preserve its biological activity.
he association of proteins, and especially enzymes, with Langmuir–Blodgett films,
using the techniques presented above, has recently been reviewed in [25], which
discusses in particular the main points of interest of such biomimetic membranes and its
applications in nanobioscience.
1.4 Biomimetic LB membrane inserting oriented proteins
The functionalisation of Langmuir–Blodgett films by association of proteins before or
after transferring the lipid leads to a random association of the protein to the biomimetic
LB membrane. One of the great challenges in the development of ordered protein–lipid
assemblies and functionalised biomimetic membranes is to control the orientation of the
associated protein, just as it is in biological membranes where the binding of the protein
on (or in) the lipidic leaflets determines its own orientation for an optimal functionality.
The building-up of organized proteo-lipidic membranes possessing properly oriented
recognition sites constitutes a promising model for further developments in biomimetic
sensing layers. It is of great interest in nanobiosciences and nanobiotechnology for many
reasons. These reasons include, (i) used in contact with a chemical (or physical) device
handling as a signal transducer, such membrane models may open a new way in the
biocatalysis investigations to access the complex functioning of biological membranes at
11
a nanometric scale; (ii) associated to microelectronic and optoelectronic devices, they
should lead to the design of new bioelectronic hybrids and the development of novel
nanobiosensors based on molecular recognition and signal transduction events of
biological systems; (iii) deposited on an ovoid scaffold and integrating ion channels or
pore proteins, they may be implied in the drug vectorisation and drug delivery [61].
In order to overcome the problem of the orientation of the protein associated with lipid
membranes in general, and Langmuir–Blodgett films in particular, several strategies have
been developed independently. These include the covalent coupling of the antigen
binding fragment of an antibody via a disulfide bridge to the polar headgroup of a linker
lipid inserted into a phospholipid monolayer [62-64], or the immobilisation of histidinecontaining proteins onto metal ion chelating lipid monolayers [65]. However, in this way,
several orientations, defined by the spatial distribution of the histidine residues on the
surface of the protein, may be obtained.
The possibility of immobilising glycosylphosphatidylinositol (GPI)-anchored proteins
may circumvent the problem of multiple orientations. The unique orientation of the
protein is then guaranteed by inserting its anchor into the phospholipidic LB films [66].
However, although this method is as biomimetic as one could hope, it only works for a
well-defined class of proteins.
With the aim of designing functionalised biomimetic membranes with unique orientation
of recognition sites, another strategy has been recently developed. The idea is to insert a
monoclonal antibody that does not inhibit biological activity in a Langmuir–Blodgett
lipid bilayer. The antibody serves as an anchor to tether the protein in an oriented position
at the membrane surface (Figure 6), both to avoid denaturation of adsorbed protein onto
lipid surfaces and to preserve biological activity over few months [67]. The membranes
obtained are polyvalent and the nature of the protein that is retained is defined by the
specificity of the inserted antibody.
In this original approach developed in our group, the functional insertion of the antibody
in the lipid membrane has been achieved by using a suitable combination of two
techniques based on molecular self-assembling properties: liposome fusion at an airbuffer interface and Langmuir-Blodgett technology. This procedure exploits the
possibility of forming a mixed monomolecular proteo-lipidic film at the air/buffer
interface using surface tension forces able to disrupt membranes of a weakly stable
protein–lipid vesicle [68, 69]. After compression, the mixed monolayer is transferred by
LB transfer [70]. The principal interest about preparing protein–lipid vesicles before
forming the interfacial monolayer is that interactions can then be set up by selfassociation between the lipid molecules and the antibodies in the vesicle membranes to
improve insertion of the antibody in the interfacial film and hence, transfer the film
without ejection of the protein. The vesicles are thus used as vectors for carrying the
antibody directly to the air/buffer interface in a lipid environment [71].
Hence, by combining these two techniques, i.e., liposomes and the LB technique, the
orientation of the antibody in the liposome membrane can be predetermined, and this
orientation will be preserved when the liposomes open at the interface. The specific
interactions initially formed in the vesicle membrane are preserved during the interfacial
vesicle disintegration and lead in turn to a preferential orientation of the antibodies in the
supported bilayer structure [72]. The organisation of the protein–lipid film is then
maintained by lateral compression of the monolayer. After immunoassociation, the target
12
protein will be retained at the surface of the bilayer membrane in a well-defined
orientation [67].
Figure 6
Structural model of functionalised LB membrane dedicated to the oriented
immobilisation of proteins.
The lipid bilayer is made from a synthetic neoglycolipid presenting highly-fluid
hydrocarbon chains. The monoclonal antibody is held in the bilayer by (i) assumed
carbohydrate interactions between the glycan moiety of the antibody and the polar
headgroups of the glycolipid, and (ii) hydrophobic interactions between the Fc fragment
of the antibody (a region rich in aliphatic residues) and the lipid moiety of the
glycolipids leaflet. In this model, the acetylcholinesterase enzyme (AChE) is associated
to the functionalised lipid bilayer after LB transfer on a solid support, by specific
immuno-recognition of the non-inhibitor antibody. This biomimetic membrane is
structurally stable and can maintain AChE activity over several months. From Godoy et
al [72].
AChE enzyme
Antibody
5 nm
10 nm
7 nm
Synthetic neoglycolipid
1.5 Potential applications of supported biomimetic LB membrane associating
protein in a well-defined orientation
From a structural point of view, the main characteristic of biomimetic membranes
associating protein in a well-defined orientation is their great stability. These
functionalised biomimetic membrane remain stable and functional for several months.
After immunoassociation of enzyme as protein model (i.e. acetylcholinesterase (AChE)
involved in the neurotransmission of the nerve influx and target of many environmental
pollutants like organophosphorus agents, Figure 6), the membrane stability allows to
retain efficient enzyme activity for a long period of time (over a period of 82 days) [67].
To our knowledge, such a high stability has never been reported previously for an
immobilised enzyme onto Langmuir-Blodgett films.
13
1.5.1 Biocatalysis investigations
Thank to such a high functional stability, which is a prerequisite for relevant
investigations, theses membranes can be used to investigate the kinetic behaviour of an
enzyme in a lipid environment on a few nanometres thick membrane model. The results
obtained with acetylcholinesterase as enzymatic model, clearly demonstrated a catalytic
enzyme behaviour characteristic of immobilised enzymes, with the marked effects of
diffusion constraints (for high substrate concentrations) in the microenvironment of the
enzyme [73]. Hence, these functional biomimetic membranes, possessing properly
oriented recognition sites by allowing an oriented binding of the enzyme at the surface of
the lipid bilayer and offering a similar topography to the one found in biological
membranes, represent a great potential for biocatalysis investigations in a general
manner, and for fundamental assessment in the field of enzymology in structured media
in particular. Most enzymes in cells are held in membranes and are found in a
phospholipid environment that is absolutely necessary for them to function correctly. The
catalytic behaviour of enzymes immobilised on a lipid bilayer is in turn fully
representative of enzyme biocatalysis of the kind observed in vivo.
1.5.2 Design of bio-optoelectronic micro/nanosensors
Biomimetic membrane associating protein in a well-defined orientation can be used to
functionalise micronic surfaces by integrating biochemical functions and to develop
biomimetic sensors exploiting sensitive layers structured on the nanoscale.
Biochemical sensors, or biosensors for short, are high-performance analytical tools,
combining the specific recognition capacity of a sensitive biological element, the
bioreceptor, with the sensitivity of the (electro-)chemical, physical, or optical sensor, the
transducer. The latter detects physicochemical changes generated by the bioreceptor upon
contact with the target substance and translates them into a measurable and interpretable
electrical signal.
The performance of a biosensor is closely linked to the properties of the sensitive layer
and the quality of its association with the transducer. Current developments follow the
marked trend toward miniaturisation of recognition systems. Molecular scale patterning
of the sensitive layer is therefore a crucial step in sensors miniaturisation.
With the aim of designing a new miniaturisable bio-optoelectronic sensor, biomimetic
membranes obtained by the Langmuir–Blodgett technique and associating
aacetylcholinesterase (AChE) in an oriented way has been combined with a highperformance optical sensor (Figure 7) [73].
This new type of sensor combines the advantages of using a biomimetic membrane as
sensitive layer with those of using screen-printed electrodes for the electrochemiluminescence reaction of luminol (ECL): (i) the membrane is ordered at the molecular
level, so there is hope for miniaturising the analysed region, (ii) it allows a functional
orientation of the enzyme at the membrane surface, something that is not always possible
with the usual methods for immobilising enzymes, (iii) the membrane, exploiting the
specific recognition properties of a non-inhibitor monoclonal antibody, is multipurpose,
whence different enzymes could be immobilised there by changing the antibody.
14
At the same time, since the detection system is triggered by hydrogen peroxide, it can be
applied to many oxidases able to detect a range of different metabolites of medical,
industrial, or pharmaceutical interest. Environmental applications are also envisaged.
Finally, the intimate contact between the different enzyme layers favours the flow of
metabolites toward the detection device, avoiding back-diffusion into the reaction
medium and increasing sensitivity. The ultrathin dimensions of the biomimetic
membrane lead to the high performance of this sensor, especially in terms of response
time.
Figure 7
Bio-optoelectronic microsensor based on biomimetic LB membrane.
This sensor has been obtained by direct transfer of a biomimetic Langmuir–Blodgett
membrane associating acetylcholinesterase (AChE) in oriented position at the surface of
a screen-printed electrode adapted for electrochemiluminescence reaction of luminol.
Briefly, acetylcholinesterase catalyses choline formation from acetylcholine present in
the reaction medium. Choline is then oxidised by choline oxidase (ChOD) immobilised
in a photopolymer of poly(vinyl alcohol) (PVA) at the surface of a screen-printed
electrode. This produces hydrogen peroxide (H2O2), which is detected in the presence
of electro-oxidised luminol by light emission focused on an optical fibre connected to
the photomultiplier tube of a light meter. From Godoy et al. [73].
Screen-printed microelectrode
+ 450 mV
vs Ag/AgCl
graphite
Choline oxidase (ChOD) included
in PVA polymer membrane
Biomimetic LB membrane
(Neoglycolipid-antibody-AChE)
Luminol
Fiberoptic
diazaquinone
+
hν
H2O2
λ max. = 425 nm
choline
acetylcholine
Working area
(0.18 cm2)
ca 10 - 20 µm
17 nm
Choline sensor
However, as it is designed, the performance of this sensor also depends on the additional
introduction of luminol during ECL measurement. Injections of soluble luminol in the
reaction medium inevitably result to a lag-time due to diffusion of this reactant through
the sensing layer to trigger ECL reaction. The possibility to insert an amphiphilic luminol
derivative directly into the lipid bilayer as support for ECL detection may give the
opportunity to develop reagentless biomimetic sensors (Figure 8). This amphiphile
derivative has been recently synthesized in our group. Its potential activity for ECL
measurement has been first investigated [74]. Its interfacial behavior and its ability to
form stable monolayers, with the final aim to directly insert it in the biomimetic
membrane has been checked [75].
At the same time, with the final aim to miniaturise the detection area of this sensor, the
enzymatic polymer membrane of few micrometers thick needs to be removed. Another
monoclonal antibody directly inserted in the biomimetic membrane may play the role of a
second anchor to fix oxidase required for ECL reaction (hydrogen peroxide generation).
This part of the work is now under investigation.
15
Finally, the interfacing of ultrathin LB membranes with a high-performance ECL device
opens a new way in the achievement of miniaturised bio-optoelectronic sensors and
further developments in macroarray systems by insertion of different biocatalytic
elements, using the diversity of the antibody recognition specificity.
Figure 8
Principle of reagentless bio-optoelectronic microsensor based on biomimetic LB
membrane. Structure of amphiphilic derivative from Tifeng et al. [74, 75].
Screen-printed
microelectrode
Biomimetic LB membrane
(Neoglycolipid - Antibodies - ChOD/AChE )
O
Amphiphilic luminol derivative
HN
HN
O
HN
OC11H23
O
O
O
O
O
OC11H23
diazaquinone
+
H2O2
ChOD
choline
hν
λ max. = 425 nm
AChE
acetylcholine
17 nm
1.5.3 Biomimetic sensors to access biogical membrane functioning
The association of Langmuir–Blodgett biomimetic membranes with high performance
optical sensors illustrates the way in which ‘natural’ patterning of the sensitive layer by
self-association of biomolecules can be combined with surface functionalisation in the
design of miniaturised bio-optoelectronic sensors.
Besides, direct interfacing of proteo-lipidic LB membrane with a chemical transducer,
allowing both recognition and transduction in a single device may be exploited to access
complex functioning of biological membrane. The main reason is the direct access of the
local environment (i.e. microenvironment) of the biological element without diffusion of
the reactants which gives a statistical and global view of the biological phenomena.
Due to the intimate contact of an LB membrane associating enzyme with the transducer,
it has been possible few years ago to detect instantaneously very low enzyme activity
[59]. The absence of diffusion constraints, related to the small enzyme amount retained
on the LB films, gives the opportunity to assess the catalytic properties (intrinsic
behaviour) of enzymes associated with LB membranes. The possibility of directly
studying through LB sensor technology, phenomena such as recognition and transducing
of molecular information, which constitutes the main biological process in natural
16
membranes, appears then attractive for further developments of models devoted to the
investigations of biological processes in a reconstituted biomimetic situation.
More recently, the intimate contact of biomimetic LB membranes associating oriented
recognition sites with a miniaturised ECL device (Figure 7), allowed to directly
investigate the enzyme kinetics thank to an efficient transduction of the biochemical
signal, like in the biological membrane [73].
From a more general standpoint, protein–lipid membranes associated with sensors can be
used to study their functional properties. This association corresponds to a biomimetic
simulation as close as one could hope to get to one of the main functions of biological
membranes, namely, the recognition and transduction of biological signals. The direct
contact between biological element and transducer allows a detailed study of its
recognition properties and the resulting physicochemical modifications, providing
information about the structure–function relations of biological membranes. If the protein
is an enzyme, one can investigate its catalytic properties in a heterogeneous medium in a
biomimetic lipid environment at the nanoscale. In particular, this type of study is relevant
in the field of nanobioscience.
1.6 Trends and perspectives
For several years, self-assembly properties of biomolecules received more and more
attention because of their ability to spontaneously organize into nanostructures, which
allows mimicking the living cell membranes.
Langmuir monolayers are membrane models exploiting the self-association properties of
amphipathic lipid molecules at the air/water interface. The main advantage with them is
the possibility of obtaining a perfectly ordered state at the water surface and then being
able to control this aggregated state by varying the imposed surface pressure. In
nanobiotechnology, the interest in developing Langmuir monolayers is double-edged. On
the one hand, they can be used to form supported lipid bilayers by transferring the
monolayer onto a solid substrate, while on the other hand they are well-suited to the study
of lipid/protein interactions or of macromolecules in general.
Langmuir-Blodgett technology is a powerful method to elaborate functionalised
biomimetic membranes. Different aspects of the biological membrane, like fluidity or
asymmetry can be preserved, but the most promising outcome resides in the possibility to
orient functional macromolecules in the bilayer structure.
By the way to be directly prepared at the surface of different kinds of solid materials, LB
membranes present some real advantages for applications in nanobiotechnology and
applied nanobiosciences. A direct association with active surfaces constitutes an
attractive opportunity for designing novel nanosensors. The intimate contact between LB
membranes and effective transducers, allowing recognition and signal transduction events
in a single device is without doubt, a very promising way for the development of
biomimetic nanosensors and minute investigations of biological processes at the
molecular level.
17
2
Biochips and microarrays: nanobiotechnology applications
2.1 Biomolecules electro-grafting
The immobilization of the biological molecules is a crucial step in the highly innovative
biochip research field since it is directly related to the biosensing performances obtained.
To date, even if a wide variety of biochips were developed, [76] no generic procedure has
merged, which could be easily applied to both protein and nucleic acid, and more
generally to interaction-based biochips. Nowadays, an obvious need exists to set-up a
flexible immobilization process, providing strong, stable and accessible binding of the
sensing-element, thus leading to sensitive and reproducible biochip performances.
Our group have recently [77] presented an immobilization strategy enabling the direct
grafting of aryl-diazonium modified non-catalytic proteins (antigens) at the surface of
screen-printed graphite electrode (SP) biochips. This approach leads to spatially resolved
grafting of proteins onto conducting surfaces. For that purpose, the biomolecules were
first modified with aniline derivatives, which were oxidized into aryl-diazonium function
prior to the electro-addressing (Figure 9).
Figure 9
Strategy for direct electro-addressing of modified antibody onto SP graphite electrode
surface. CMA: 4-CarboxyMethylAniline, DDC: N,N'-dicyclohexyl-carbodiimide.
This technique is based on the particular electrochemical property of aryl-diazonium salts
[78]. These molecules could be electro-addressed at the surface of a polarized electrode,
leading to the formation of a covalent C-X (X being the electrode material) bond between
the aryl group and the electrode (Figure 10). This electrochemical-grafting property of
aryl-diazonium derivatives was previously confirmed on a large variety of conductive
and semi-conductive material such as carbon, metal, silicon, diamond and recently on
ITO electrodes [79-83].
We lately demonstrated, in a proof of concept study, the usefulness of this technique for
the electro-addressed immobilization of biomolecules as different as antibody, nucleic
acid and enzyme [84]. The potentialities of the electro-addressing chemistry are
illustrated through different biosensing architecture – i.e. oligonucleotide based assay,
18
capture immunoassay and sandwich immunoassay – all involving a chemiluminescent
detection system using horseradish peroxidase as label (Figure 11).
Figure 10 The aniline derivative electro-addressing mechanism: i) diazotation of the aniline
derivative, ii) electro-reduction of diazonium at a conductive electrode surface, iii)
covalent grafting to the carbon electrode surface via a C-C bound.
R
R
R
+e-
i
NH2
R
N2
ii
iii
N
N
The first demonstration of an analytical application of the electro-addressing of aryldiazonium modified biomolecule is based on oligonucleotide functionalized biochip. To
our knowledge, only a few works deals with the direct covalent binding of
oligonucleotides on conducting material [82, 85]. Here, a 20mer sequence from a "hot
spot" of the exon 8 of the p53 tumor suppressor gene [83] was functionalized with 4aminobenzylamine (4-ABA), electro-addressed and used as stationary phase probe
sequence for hybridization testing of biotinylated target sequence (Figure 11-a). The
probe sequence was here functionalized at its 5’-end with 4-ABA to provide an orientated
grafting. The probe surface density was estimated using XPS experiment (S.I) and was
found to be 3.75 10-13 molecules/cm2. This result compares well with similar XPS
experiments for surface coverage determination on gold substrate [86].
Similar studies were systematically performed with 4-carboxyaniline (4-CMA) modified
proteins as addressed biomolecules. First, rabbit immunoglobulins (IgG) were used as
immobilized antigens and involved in the detection of rheumatoid factor (RF) – i.e. a
family of human antibodies largely involved in rheumatoid diseases [87] and which the
presence could be characterized by an anti-rabbit IgG activity of the serum. The assay, a
capture format (Figure 11-b), is not considered as a sandwich assay since the
immobilized rabbit IgGs are not used as active antibodies but as capture antigens.
Nevertheless, the success of the assay is determined by the accessibility of the different
epitopes of the immobilized IgG toward the numerous paratopes of the polyclonal human
sera antibodies [88]. Different human serum samples containing known concentrations
of rheumatoid factor were incubated on the rabbit IgG modified biochip surface. A clear
correlation between the measured chemiluminescent signal and the RF value in the serum
samples was found, allowing the detection of RF in the 5.3-485 IU/ml range with an
acceptable accuracy when compared to previous works (standard Auraflex® ELISA test
and [88]).
For the immobilization technique to be fully demonstrated as useful for biosensing, a
second immunochemical application based on a sandwich immunoassay procedure has
been demonstrated. It involves the recognition properties of electro-addressed anti-human
IgGs and the binding properties of the grafted antibodies are directly implicated in the
detection process of the antigen in solution, here a human IgG (Figure 11-c). Thus, every
loss of integrity of the immobilized antibodies would have a dramatic effect on the
recognition event. In this case, we were able to demonstrate that the immobilized antihuman IgG antibodies could be successfully involved in the recognition of the free target
19
protein, evidencing that the proposed immobilization procedure maintains a convenient
structural conformation of the grafted biomolecules, allowing them to be implicated in a
recognition process.
Figure 11 Schematic representations of the different proofs of concept using the electro-addressed
immobilisation of (a) a probe DNA sequence, (b) an immunoglobulin antigen and (c) an
active anti-human antibody.
2.2 Gold nanotexturation for the on-chip chemiluminescent enhancement
Our group recently described an original method for the enhancement of
chemiluminescent (CL) on-chip detection of protein and oligonucleotide [89]. This
enhancement is based on the electro-deposition of a gold nanostructured layer onto a
screen-printed (SP) carbon microarray, prior to the immobilization of biomolecules
through the diazonium adduct electro-deposition process. Morphological studies of the
Au layer (optical and atomic force microscopy) show that the metal film is composed of
nanostructured (rms 16.5 nm) 800 nm diameter particles covering the entire graphite
surface and yielding a high surface area (Figure 12). Using these modified SP
microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific
20
antigen (PSA) immuno-detection and p53 oligonucleotide detection, respectively. These
enhancements were associated with three different phenomena: an enhancement of the
catalyzed chemiluminescent reaction by the gold surface, an increase of the specific
surface area for immobilization of the probe biomolecules, and an opposite quenching
effect due to the overlapping of the gold absorption and the CL emission peaks. For the
free PSA and target oligonucleotide detection, enhanced performances were thus
obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.
Figure 12 Atomic force microscopy (NT-MDT, tapping mode) images of a) a bare SP electrode
and b-d) different magnifications of an Au*SP electrode. The individual 800 nm
particle roughness is 16.5 nm (rms).
2.3 Direct polymer modification with biomolecules
Since the emergence of the miniaturization concept [90] and the need for improved
biomedical devices [91], polymers have become very popular materials for the
development of micro and nano fabricated biological systems [92]. The early stage was
focused on glass or silicon technology, particularly in the field of lab-on-chip devices or
surface patterning, but various polymers (for example PDMS, PMMA, PTFE, PS) [9395] gained rapid interest because of their availability, their cost and the broad range of
chemical reactivity allowing an easy and customisable biomolecules grafting [96].
Following this evolution, PDMS appeared rapidly as a powerful material for rapid
prototyping and was used for many different applications among which immunoassays
21
[97, 98], microfluidics [99, 100], surface patterning [101, 102] and electrophoresis [103].
Indeed, PDMS exhibits several attractive properties required for the development and
prototyping of micro fabricated tools [104] such as low cost, ease to use in standard
academic laboratory conditions, good optical transparency, gas permeability, biological
compatibility due to its low toxicity and finally very low pattern resolution when
moulded on a substrate [105, 106]. However, these advantages are counter balanced by a
very high hydrophobicity and a chemical inertness of the polymer which triggered the
need for efficient surface modification procedures. Most of the time, a strong oxidative
step (UV irradiation, UV/ozone treatment or plasma exposure) [107] is applied to
generate a hydrophilic glass like surface allowing well known silanization coupling
chemistry with biomolecules [108]. All these steps remain time, energy and chemicals
consuming and impart some interesting qualities of the PDMS.
Figure 13 Overview of the “Macromolecules to PDMS transfer” technique highlighting the 4
main steps leading to the achievement of protein spots directly and entrapped at the
PDMS interface. SEM image of a proteins microarray at the surface of the PDMS.
In order to overcome this surface modification issue, our group developed in the last
three years another approach for the direct PDMS functionalization (Figure 13). The
method, now called “Macromolecules to PDMS transfer”, allows direct PDMS surface
modification with active proteins [109] or modified DNA [110] for the development of
biochips. This procedure relies on the ability of the PDMS polymer to entrap
macromolecules at its surface while the polymerization process occurs. Briefly,
macromolecules spots are patterned using a piezo arrayer on a 3D mould (glass or
Teflon) before being covered with liquid PDMS. Then, curing at high temperature
rendered the polymer elastomeric enabling its separation from the mould. Once peeled
off, the polymer exhibits active protein spots at the PDMS air interface. The main
advantage of this concept is to be able to combine both the macromolecule
22
immobilization, without the need of additional chemicals, and the easy 3D structures
achievement according to the initial mould structure.
Numerous applications were already described using this method and we are presenting
herein a focus on the implementation of the technique showing how different proteins can
be successfully immobilized and used for (i) the development of sensitive sandwich
immunoassay for C-reactive protein (CRP) detection, (ii) the characterisation of patient
sera according to rheumatoid factor (RF) level and finally (iii) demonstrate how the
procedure can be used to easily and rapidly produce fibronectin based cell culture arrays
(Figure 14).
Figure 14 Schematic representation of the sandwich CRP assay (A), the RF capture assay (B) and
the cell culture biochip (C), based on the “Macromolecules to PDMS transfer”
procedure.
First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal
anti-CRP antibodies was demonstrated for sepsis diagnosis. The preserved integrity of the
immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP
in human sera (LOD=12.5µg/L, detection ranging over two decades). Then, rheumatoid
arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit
immunoglobulins immobilisation was shown to allow the detection of specific antibodies
in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally,
the “Macromolecules to PDMS transfer” procedure was used to easily and rapidly
produce fibronectin based cell culture arrays. The successful attachment of HeLa and
BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin
and vinculin (Figure 15).
The extension of such localised cell culture is now under investigation for the
characterisation of nanostructure/cells interactions and mechanisms.
23
Figure 15 Optical microscope images of HeLA cells cultured onto 150µm fibronectin spots. Phase
contrast image (A), FITC fluorescent image after vinculin immuno-staining (B), Alexa
Fluor® 546 fluorescent image after filamentous actin staining (C) and DAPI nuclei
staining fluorescent image (D).
Acknowledgements
This work was partially supported by (i) French minister MENRT (Ministère de
l’éducation Nationale de la Recherche et Technologie) for Doctoral fellowship of Miss
Stéphanie Godoy-Violot (2001-2004), (ii) CNRS (Centre National de la Recherche
Scientifique), department of chemical science, section 12, (N°06-209) for post-doctoral
fellowship of Dr. Tifeng Jiao (October 2006 – September 2007) and (ii) Centre de
Compétences en nanosciences de la région Rhône-Alpes, C'Nano Rhône-Alpes for
Brewster Angle Microscope acquisition.
References
1
2
3
4
5
6
7
8
Reimhult, E. and Kumar, K., (2008) 'Membrane biosensor platforms using nanoand microporous supports', Trends Biotechnol., Vol. 26, No 2, pp. 82-89.
Martin, D.K. (2007) Nanobiotetechnology of biomimetic membranes, Springer
Science+Business, LLC, New York.
Richter, R.P., Berat, R. and Brisson, A.R., (2006) 'Formation of Solid-Supported
Lipid Bilayers: An Integrated View', Langmuir, Vol. 22, No 8, pp. 3497-3505.
Girard-Egrot, A.P. and Blum, L.J. (2007) Langmuir-Blodgett technique for synthesis
of biomimetic lipid membranes, in Fundamental biomedical technologies, D.K.
Martin, Editor. Springer Science+Business Media, LLC: New York.
Rossi, C. and Chopineau, J., (2007) 'Biomimetic tethered lipid membranes designed
for membrane-protein interaction studies ', Eur. Biophys. J., Vol. 36, No 8, pp. 955965.
Vollhardt, D., (2002) 'Supramolecular organisation in monolayers at the air/water
interface', Mat. Sci. Engin. C, Vol. 22, No 2, pp. 121-127.
Leblanc, R.M., (2006) 'Molecular recognition at Langmuir monolayers', Curr. Opin.
Chem. Biol., Vol. 10, No 6, pp. 529-536.
El Kirat, K., Besson, F., Prigent, A.-F., Chauvet, J.-P. and Roux, B., (2002) 'Role of
Calcium and Membrane Organization on Phospholipase D Localization and
Activity. Copetition Between a Soluble and an Insoluble Substrate.' J. Biol. Chem.,
Vol. 277, No 24, pp. 21231-21236.
24
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Girard-Egrot, A., Chauvet, J.-P., Gillet, G. and Moradi-Améli, M., (2004) 'Specific
Interaction of the Antiapoptotic Protein Nr-13 with Phospholipid Monolayers is
Prevented by the BH3 Domain of Bax', J. Mol. Biol., Vol. 335, No 1, pp. 321-331.
Blodgett, K.B., (1935) 'Films Built by Depositing Successive Monomolecular
Layers on a Solid Surface', J. Am. Chem. Soc., Vol. 57, No 6, pp. 1007-1022.
Blodgett, K.B. and Langmuir, I., (1937) 'Built-Up Films of Barium Stearate and
Their Optical Properties', Phys. Rev. A, Vol. 51, No 11, pp. 964-982.
Morelis, R.M., Girard-Egrot, A.P. and Coulet, P.R., (1993) 'Dependence of
Langmuir-Blodgett film quality on fatty acid monolayer integrity. 1. Nucleation
crystal growth avoidance in the monolayer through the optimized compression
procedure', Langmuir, Vol. 9, No 11, pp. 3101-3106.
Girard-Egrot, A.P., Morelis, R.M. and Coulet, P.R., (1993) 'Dependence of
Langmuir-Blodgett film quality on fatty acid monolayer integrity. 2. Crucial effect
of the removal rate of monolayer during Langmuir-Blodgett film deposition',
Langmuir, Vol. 9, No 11, pp. 3107-3110.
Girard-Egrot, A.P., Morelis, R.M. and Coulet, P.R., (1996) 'Direct Influence of the
Interaction between the First Layer and a Hydrophilic Substrate on the Transition
from Y- to Z-Type Transfer during Deposition of Phospholipid Langmuir-Blodgett
Films', Langmuir, Vol. 12, No 3, pp. 778-783.
Brockman, H., (1999) 'Lipid monolayers: why use half a membrane to characterize
protein-membrane interactions?' Current Opinion in Structural Biology, Vol. 9, No
4, pp. 438-443.
Maget-Dana, R., (1999) 'The monolayer technique: a potent tool for studying the
interfacial properties of antimicrobial and membrane-lytic peptides and their
interactions with lipid membranes', Biochim. Biophys. Acta, Vol. 1462, No 1-2, pp.
109-140.
Krishnakumari, V. and Nagaraj, R., (2008) 'Interaction of antibacterial peptides
spanning the carboxy-terminal region of human [beta]-defensins 1-3 with
phospholipids at the air-water interface and inner membrane of E. coli', Peptides,
Vol. 29, No 1, pp. 7-14.
Dhathathreyan, A. and Steinem, C., (2008) 'Interactions of laminin peptides with
phospholipids in Langmuir films and vesicles', Chem. Phys. Lett., Vol. 464, No 4-6,
pp. 226-229.
Fontvila, O., Mestres, C., Muñoz, M., Haro, I., Alsina, M.A. and Pujol, M., (2008)
'Surface behaviour and peptide-lipid interactions of the E1(3-17)R and E1(3-17)G
peptides from E1 capside protein of GBV-C/HGV virus', Coll. Surf. A, Vol. 321, No
1-3, pp. 175-180.
Henon, S. and Meunier, J., (1991) 'Microscope at the Brewster angle: Direct
observation of first-order phase transitions in monolayers', Rev. Sci. Instrum., Vol.
62, No 4, pp. 936-939.
Hoenig, D. and Moebius, D., (1991) 'Direct visualization of monolayers at the airwater interface by Brewster angle microscopy', J. Phys. Chem., Vol. 95, No 12, pp.
4590-4592.
Vollhardt, D. and Fainerman, V.B., (2000) 'Penetration of dissolved amphiphiles
into two-dimensional aggregating lipid monolayers', Adv. Colloid Interface Sci.,
Vol. 86, No 1-2, pp. 103-151.
Vollhardt, D., (1996) 'Morphology and phase behavior of monolayers', Adv. Colloid
Interface Sci., Vol. 64, No pp. 143-171.
25
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Honnorat, J., Byk, T., Kusters, I., Aguera, M., Ricard, D., Rogemond, V., Quach, T.,
Aunis, D., Sobel, A., Mattei, M.-G., Kolattukudy, P., Belin, M.-F. and Antoine, J.C.,
(1999) 'Ulip/CRMP proteins are recognized by autoantibodies in paraneoplastic
neurological syndromes', Eur. J. Neurosci., Vol. 11, No 12, pp. 4226-4232.
Girard-Egrot, A.P., Godoy, S. and Blum, L.J., (2005) 'Enzyme association with
lipidic Langmuir-Blodgett films: Interests and applications in nanobioscience', Adv.
Colloid Interface Sci., Vol. 116, No 1-3, pp. 205-225.
Choi, J.-W., Min, J., Jung, J.-W., Rhee, H.-W. and Lee, W.H., (1998) 'Fiber-optic
biosensor for the detection of organophosphorus compounds using AChEimmobilized viologen LB films', Thin Solid Films, Vol. 327-329, No pp. 676-680.
Moriizumi, T., (1988) 'Langmuir-Blodgett films as chemical sensors', Thin Solid
Films, Vol. 160, No 1-2, pp. 413-429.
Arisawa, S. and Yamamoto, R., (1992) 'Quantitative characterization of enzymes
adsorbed on to Langmuir-Blodgett films and the application to a urea sensor', Thin
Solid Films, Vol. 210-211, No Part 2, pp. 443.
Yasuzawa, M., Hashimoto, M., Fujii, S., Kunugi, A. and Nakaya, T., (2000)
'Preparation of glucose sensors using the Langmuir-Blodgett technique', Sens.
Actuators, B, Vol. 65, No 1-3, pp. 241-243.
Zhu, D.G., Petty, M.C., Ancelin, H. and Yarwood, J., (1989) 'On the formation of
Langmuir-Blodgett films containing enzymes', Thin Solid Films, Vol. 176, No 1, pp.
151-156.
Fromherz, P., (1971) 'A new technique for investigating lipid protein films',
Biochim. Biophys. Acta, Vol. 225, No 2, pp. 382-387.
Verger, R. and Pattus, F., (1982) 'Lipid-protein interactions in monolayers', Chem.
Phys. Lipids., Vol. 30, No 2-3, pp. 189-227.
Sriyudthsak, M., Yamagishi, H. and Moriizumi, T., (1988) 'Enzyme-immobilized
Langmuir-Blodgett film for a biosensor', Thin Solid Films, Vol. 160, No 1-2, pp.
463-469.
Miyauchi, S., Arisawa, S., Arise, T. and Yamamoto, R., (1989) 'Study on the
concentration of an enzyme immobilized by Langmuir-Blodgett films', Thin Solid
Films, Vol. 180, No 1-2, pp. 293-298.
Ancelin, H., Zhu, D.G., Petty, M.C. and Yarwood, J., (1990) 'Infrared spectroscopic
studies of molecular structure, ordering, and interactions in enzyme-containing
Langmuir-Blodgett films', Langmuir, Vol. 6, No 6, pp. 1068-1070.
Arisawa, S., Arise, T. and Yamamoto, R., (1992) 'Concentration of enzymes
adsorbed onto Langmuir films and characteristics of a urea sensor', Thin Solid Films,
Vol. 209, No 2, pp. 259-263.
Fujiwara, I., Ohnishi, M. and Seto, J., (1992) 'Atomic force microscopy study of
protein-incorporating Langmuir-Blodgett films', Langmuir, Vol. 8, No 9, pp. 22192222.
Fiol, C., Alexandre, S., Delpire, N., Valleton, J.M. and Paris, E., (1992) 'Molecular
resolution images of enzyme-containing Langmuir-Blodgett films', Thin Solid Films,
Vol. 215, No 1, pp. 88-93.
Fiol, C., Valleton, J.-M., Delpire, N., Barbey, G., Barraud, A. and Ruaudel-Teixier,
A., (1992) 'Elaboration of a glucose biosensor based on Langmuir-Blodgett
technology', Thin Solid Films, Vol. 210-211, No Part 2, pp. 489-491.
Rosilio, V., Boissonnade, M.-M., Zhang, J., Jiang, L. and Baszkin, A., (1997)
'Penetration of Glucose Oxidase into Organized Phospholipid Monolayers Spread at
the Solution/Air Interface', Langmuir, Vol. 13, No 17, pp. 4669-4675.
26
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
Tang, F.Q., Li, J.R., Zhang, L. and Jiang, L., (1992) 'Improvement of enzymatic
activity and lifetime of Langmuir-Blodgett films by using submicron SiO2 particles',
Biosens. Bioelectron., Vol. 7, No 7, pp. 503-507.
Barraud, A., Perrot, H., Billard, V., Martelet, C. and Therasse, J., (1993) 'Study of
immunoglobulin G thin layers obtained by the Langmuir-Blodgett method:
application to immunosensors', Biosens. Bioelectron., Vol. 8, No 1, pp. 39-48.
Ramsden, J.J., Bachmanova, G.I. and Archakov, A.I., (1996) 'Immobilization of
proteins to lipid bilayers', Biosens. Bioelectron., Vol. 11, No 5, pp. 523-528.
Heckl, W.M., Zaba, B.N. and Möhwald, H., (1987) 'Interactions of cytochromes b5
and c with phospholipid monolayers', Biochim. Biophys. Acta, Vol. 903, No 1, pp.
166-176.
Wan, K., Chovelon, J.M. and Jaffrezic-Renault, N., (2000) 'Enzyme-octadecylamine
Langmuir-Blodgett membranes for ENFET biosensors', Talanta, Vol. 52, No 4, pp.
663-670.
Girard-Egrot, A.P., Morelis, R.M. and Coulet, P.R., (1997) 'Choline Oxidase
Associated with Behenic Acid LB Films. Reorganization of Enzyme-Lipid
Association under the Conditions of Activity Detection', Langmuir, Vol. 13, No 24,
pp. 6540-6546.
Singhal, R., Gambhir, A., Pandey, M.K., Annapoorni, S. and Malhotra, B.D., (2002)
'Immobilization of urease on poly(N-vinyl carbazole)/stearic acid LangmuirBlodgett films for application to urea biosensor', Biosens. Bioelectron., Vol. 17, No
8, pp. 697-703.
Lee, S., Anzai, J.-i. and Osa, T., (1993) 'Enzyme-modified Langmuir-Blodgett
membranes in glucose electrodes based on avidin-biotin interaction', Sens.
Actuators, B, Vol. 12, No 2, pp. 153-158.
Choi, J.-W., Kim, Y.-K., Lee, I.-H., Min, J. and Lee, W.H., (2001) 'Optical
organophosphorus biosensor consisting of acetylcholinesterase/viologen hetero
Langmuir-Blodgett film', Biosens. Bioelectron., Vol. 16, No 9-12, pp. 937-943.
Caseli, L., Zaniquelli, M.E.D., Furriel, R.P.M. and Leone, F.A., (2002) 'Enzymatic
activity of alkaline phosphatase adsorbed on dimyristoylphosphatidic acid
Langmuir-Blodgett films', Coll. Surf. B, Vol. 25, No 2, pp. 119-128.
Marron-Brignone, L., Morélis, R.M., Blum, L.J. and Coulet, P.R., (1996) 'Behaviour
of firefly luciferase associated with Langmuir-Blodgett films', Thin Solid Films, Vol.
284-285, No pp. 784-788.
Anzai, J.-i., Lee, S. and Osa, T., (1989) 'Enzyme-immobilized Langmuir-Blodgett
membranes for biosensor application. Use of highly branched polyethyleneimine as
a spacer for immobilizing α-chymotrypsin and urease', Die Makromolekulare
Chemie, Rapid Communications, Vol. 10, No 4, pp. 167-170.
Tatsuma, T., Tsuzuki, H., Okawa, Y., Yoshida, S. and Watanabe, T., (1991)
'Bifunctional Langmuir-Blodgett film for enzyme immobilization and amperometric
biosensor sensitization', Thin Solid Films, Vol. 202, No 1, pp. 145-150.
Tsuzuki, H., Watanabe, T., Okawa, Y., Yoshida, S., Yano, S., Koumoto, K.,
Komiyama, M. and Nihei, Y., (1988) 'A Novel Glucose Sensor with a Glucose
Oxidase Monolayer Immobilized by the Langmuir–Blodgett Technique', Chem.
Lett., Vol. 17, No 8, pp. 1265-1268.
Zhang, A., Hou, Y., Jaffrezic-Renault, N., Wan, J., Soldatkin, A. and Chovelon, J.M., (2002) 'Mixed urease/amphiphile LB films and their application for biosensor
development', Bioelectrochemistry, Vol. 56, No 1-2, pp. 157-158.
27
56
57
58
59
60
61
62
63
64
65
66
67
68
69
Hou, Y., Tlili, C., Jaffrezic-Renault, N., Zhang, A., Martelet, C., Ponsonnet, L.,
Errachid, A., samitier, J. and Bausells, J., (2004) 'Study of mixed Langmuir-Blodgett
films of immunoglobulin G/amphiphile and their application for immunosensor
engineering', Biosens. Bioelectron., Vol. 20, No 6, pp. 1126-1133.
Girard-Egrot, A.P., Morelis, R.M. and Coulet, P.R., (1998) 'Influence of lipidic
matrix and structural lipidic reorganization on choline oxidase activity retained in
LB films', Langmuir, Vol. 14, No 2, pp. 476-482.
Girard-Egrot, A.P., Morélis, R.M. and Coulet, P.R., (1997) 'Bioactive nanostructure
with glutamate dehydrogenase associated with LB films: protecting role of the
enzyme molecules on the structural lipidic organization', Thin Solid Films, Vol. 292,
No 1-2, pp. 282-289.
Girard-Egrot, A.P., Morelis, R.M. and Coulet, P.R., (1998) 'Direct
bioelectrochemical monitoring of choline oxidase kinetic behaviour in LangmuirBlodgett nanostructures', Bioelectrochemistry and Bioenergetics, Vol. 46, No 1, pp.
39-44.
Berzina, T.S., Piras, L. and Troitsky, V.I., (1998) 'Study of horseradish peroxidase
activity in alternate-layer Langmuir-Blodgett films', Thin Solid Films, Vol. 327-329,
No pp. 621-626.
Battle, A.R., Valenzuela, S.M., Mechler, A., Nichols, R.J., Praporski, S., di Maio,
I.L., Islam, H., Girard-Egrot, A.P., Cornell, B.A., Prashar, J., Caruso, F., Martin,
L.L. and Martin, D.K., (2009) 'Novel Engineered Ion Channel Provides Controllable
Ion Permeability for Polyelectrolyte Microcapsules Coated with a Lipid Membrane',
Adv. Func. Mater., Vol. 19, No 2, pp. 201-208.
Vikholm, I. and Albers, W.M., (1998) 'Oriented Immobilization of Antibodies for
Immunosensing', Langmuir, Vol. 14, No 14, pp. 3865-3872.
Ihalainen, P. and Peltonen, J., (2002) 'Covalent Immobilization of Antibody
Fragments onto Langmuir-Schaefer Binary Monolayers Chemisorbed on Gold',
Langmuir, Vol. 18, No 12, pp. 4953-4962.
Vikholm, I., Viitala, T., Albers, W.M. and Peltonen, J., (1999) 'Highly efficient
immobilisation of antibody fragments to functionalised lipid monolayers', Biochim.
Biophys. Acta, Vol. 1421, No 1, pp. 39-52.
Kent, M.S., Yim, H., Sasaki, D.Y., Majewski, J., Smith, G.S., Shin, K., Satija, S.
and Ocko, B.M., (2002) 'Segment Concentration Profile of Myoglobin Adsorbed to
Metal Ion Chelating Lipid Monolayers at the Air-Water Interface by Neutron
Reflection', Langmuir, Vol. 18, No 9, pp. 3754-3757.
Caseli, L., Furriel, R.P.M., de Andrade, J.F., Leone, F.A. and Zaniquelli, M.E.D.,
(2004) 'Surface density as a significant parameter for the enzymatic activity of two
forms of alkaline phosphatase immobilized on phospholipid Langmuir-Blodgett
films', J. Colloid Interface Sci., Vol. 275, No 1, pp. 123-130.
Godoy, S., Chauvet, J.-P., Boullanger, P., Blum, L.J. and Girard-Egrot, A.P., (2003)
'New Functional Proteo-glycolipidic Molecular Assembly for Biocatalysis Analysis
of an Immobilized Enzyme in a Biomimetic Nanostructure', Langmuir, Vol. 19, No
13, pp. 5448-5456.
Girard-Egrot, A.P., Chauvet, J.P., Boullanger, P. and Coulet, P.R., (2001)
'Glycolipid and monoclonal immunoglobulin-glycolipidic liposomes spread onto
high ionic strength buffers: Evidence for a true monolayer formation', Langmuir,
Vol. 17, No 4, pp. 1200-1208.
Girard-Egrot, A.P., Godoy, S., Chauvet, J.P., Boullanger, P. and Coulet, P.R., (2003)
'Preferential orientation of an immunoglobulin in a glycolipid monolayer controlled
by the disintegration kinetics of proteo-lipidic vesicles spread at an air-buffer
interface', Biochim. Biophys. Acta, Vol. 1617, No 1-2, pp. 39-51.
28
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
Girard-Egrot, A.P., Chauvet, J.P., Boullanger, P. and Coulet, P.R., (2002) 'IgG1glycolipidic LB films obtained by vertical deposition of an interfacial film formed
through proteo-liposome spreading at the air/water interface', Coll. Surf. B, Vol. 23,
No 4, pp. 319-325.
Girard-Egrot, A.P., Morelis, R.M., Boullanger, P. and Coulet, P.R., (2000)
'Immunological proteo-glycolipidic interfacial film obtained from spreading of
liposomes including ascitic fluid', Coll. Surf. B, Vol. 18, No 2, pp. 125-135.
Godoy, S., Violot, S., Boullanger, P., Bouchu, M.-N., Leca-Bouvier, B.D., Blum,
L.J. and Girard-Egrot, A.P., (2005) 'Kinetics Study of Bungarus fasciatus Venom
Acetylcholinesterase Immobilised on a Langmuir-Blodgett Proteo-Glycolipidic
Bilayer', ChemBioChem, Vol. 6, No 2, pp. 395-404.
Godoy, S., Leca-Bouvier, B., Boullanger, P., Blum, L.J. and Girard-Egrot, A.P.,
(2005)
'Electrochemiluminescent
detection
of
acetylcholine
using
acetylcholinesterase immobilized in a biomimetic Langmuir-Blodgett nanostructure',
Sens. Actuators, B, Vol. 107, No 1, pp. 82-87.
Jiao, T., Leca-Bouvier, B.D., Boullanger, P., Blum, L.J. and Girard-Egrot, A.P.,
(2008) 'Electrochemiluminescent detection of hydrogen peroxide using amphiphilic
luminol derivatives in solution', Coll. Surf. A, Vol. 321, No 1-3, pp. 143-146.
Jiao, T., Leca-Bouvier, B.D., Boullanger, P., Blum, L.J. and Girard-Egrot, A.P.,
(2008) 'Phase behavior and optical investigation of two synthetic luminol derivatives
and glycolipid mixed monolayers at the air-water interface', Coll. Surf. A, Vol. 321,
No 1-3, pp. 137-142.
Stoll, D., Templin, M.F., Bachmann, J. and Joos, T.O., (2005) 'Protein microarrays:
Applications and future challenges', Curr. Opin. Drug. Disc. Dev., Vol. 8, No 2, pp.
239-252.
Corgier, B.P., Marquette, C.A. and Blum, L.J., (2005) 'Diazonium-Protein Adducts
for Graphite Electrode Microarrays Modification: Direct and Addressed
Electrochemical Immobilization', J. Am. Chem. Soc., Vol. 127, No 51, pp. 1832818332.
Delamar, M., Hitmi, R., Pinson, J. and Saveant, J.M., (1992) 'Covalent Modification
Of Carbon Surfaces By Grafting Of Functionalized Aryl Radicals Produced From
Electrochemical Reduction Of Diazonium Salts', J. Am. Chem. Soc., Vol. 114, No
14, pp. 5883-5884.
Pinson, J. and Podvorica, F., (2005) 'Attachment of organic layers to conductive or
semiconductive surfaces by reduction of diazonium salts', Chem. Soc. Rev., Vol. 34,
No 5, pp. 429-439.
Maldonado, S., Smith, T.J., Williams, R.D., Morin, S., Barton, E. and Stevenson,
K.J., (2006) 'Surface modification of indium tin oxide via electrochemical reduction
of aryldiazonium cations', Langmuir, Vol. 22, No 6, pp. 2884-2891.
Brooksby, P.A. and Downard, A.J., (2004) 'Electrochemical and Atomic Force
Microscopy Study of Carbon Surface Modification via Diazonium Reduction in
Aqueous and Acetonitrile Solutions', Langmuir, Vol. 20, No 12, pp. 5038-5045.
Ruffien, A., Dequaire, M. and Brossier, P., (2003) 'Covalent immobilization of
oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for
viral DNA sensing', Chem. Comm., Vol. 9, No 7, pp. 912-913.
Fujiki, T., Haraoka, S., Yoshioka, S., Ohshima, K., Iwashita, A. and Kikuchi, M.,
(2002) 'p53 Gene mutation and genetic instability in superficial multifocal
esophageal squamous cell carcinoma', Int. J. Oncol., Vol. 20, No 4, pp. 669-679.
Corgier, B.P., Laurent, A., Perriat, P., Blum, L.J. and Marquette, C.A., (2007) 'A
Versatile Method for Direct and Covalent Immobilization of DNA and Proteins on
Biochips', Angew. Chem. Int. Ed. Engl., Vol. 46, No 22, pp. 4108-4110.
29
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
Marquette, C.A., Lawrence, M.F. and Blum, L.J., (2006) 'DNA Covalent
Immobilization onto Screen-Printed Electrode Networks for Direct Label-Free
Hybridization Detection of p53 Sequences', Anal. Chem., Vol. 78, No 3, pp. 959964.
Petrovykh, D.Y., Kimura-Suda, H., Tarlov, M.J. and Whitman, L.J., (2004)
'Quantitative characterization of DNA films by X-ray photoelectron spectroscopy',
Langmuir, Vol. 20, No 2, pp. 429-440.
Moore, T.L. and Dorner, R.W., (1993) 'Rheumatoid factors', Clin. Biochem., Vol.
26, No 2, pp. 75-78.
Cretich, M., Pirri, G., Damin, F., Solinas, I. and Chiari, M., (2004) 'A new
polymeric coating for protein microarrays', Anal. Biochem., Vol. 332, No 1, pp. 6774.
Corgier, B.P., Li, F., Blum, L.J. and Marquette, C.A., (2007) 'On-Chip
Chemiluminescent Signal Enhancement Using Nanostructured Gold-Modified
Carbon Microarrays', Langmuir, Vol. 23, No 16, pp. 8619-8623.
Manz, A., Graber, N. and Widmer, H.M., (1990) 'Miniaturized total chemical
analysis systems: A novel concept for chemical sensing', Sens. Actuators, B, Vol. 1,
No 1-6, pp. 244-248.
Angenendt, P., (2005) 'Progress in protein and antibody microarray technology',
Drug Discov. Today, Vol. 10, No 7, pp. 503-511.
Yager, P., Edwards, T., Fu, E., Helton, K., Nelson, K., Tam, M.R. and Weigl, B.H.,
(2006) 'Microfluidic diagnostic technologies for global public health', Nature, Vol.
442, No 7101, pp. 412-418.
Becker, H. and Locascio, L.E., (2002) 'Polymer microfluidic devices', Talanta, Vol.
56, No 2, pp. 267-287.
Guber, A.E., Heckele, M., Herrmann, D., Muslija, A., Saile, V., Eichhorn, L.,
Gietzelt, T., Hoffmann, W., Hauser, P.C., Tanyanyiwa, J., Gerlach, A., Gottschlich,
N. and Knebel, G., (2004) 'Microfluidic lab-on-a-chip systems based on polymers:
fabrication and application', Chem. Eng. J., Vol. 101, No 1-3, pp. 447-453.
Becker, H. and Gärtner, C., (2008) 'Polymer microfabrication technologies for
microfluidic systems', Anal. Bioanal. Chem., Vol. 390, No 1, pp. 89-111.
96. Goddard, J.M. and Hotchkiss, J.H., (2007) 'Polymer surface modification for
the attachment of bioactive compounds', Prog. Polym. Sci., Vol. 32, No 7, pp. 698725.
Eteshola, E. and Leckband, D., (2001) 'Development and characterization of an
ELISA assay in PDMS microfluidic channels', Sens. Actuators, B, Vol. 72, No 2, pp.
129-133.
Cesaro-Tadic, S., Dernick, G., Juncker, D., Buurman, G., Kropshofer, H., Michel,
B., Fattinger, C. and Delamarche, E., (2004) 'High-sensitivity miniaturized
immunoassays for tumor necrosis factor [small alpha] using microfluidic systems',
Lab. Chip., Vol. 4, No 6, pp. 563-569.
Sia, S.K. and Whitesides, G.M., (2003) 'Microfluidic devices fabricated in
Poly(dimethylsiloxane) for biological studies', Electrophoresis, Vol. 24, No 21, pp.
3563-3576.
Duffy, D.C., McDonald, J.C., Schueller, O.J.A. and Whitesides, G.M., (1998) 'Rapid
Prototyping of Microfluidic Systems in Poly(dimethylsiloxane)', Anal. Chem., Vol.
70, No pp. 4974-4964.
Delamarche, E., Donzel, C., Kamounah, F.S., Wolf, H., Geissler, M., Stutz, R.,
Schmidt-Winkel, P., Michel, B., Mathieu, H.J. and Schaumburg, K., (2003)
'Microcontact Printing Using Poly(dimethylsiloxane) Stamps Hydrophilized by
Poly(ethylene oxide) Silanes', Langmuir, Vol. 19, No 21, pp. 8749-8758.
30
102 Bernard, A., Renault, J.P., Michel, B., Bosshard, H.R. and Delamarche, E., (2000)
'Microcontact Printing of Proteins', Adv. Mater., Vol. 12, No 14, pp. 1067-1070.
103 Dodge, A., Brunet, E., Chen, S., Goulpeau, J., Labas, V., Vinh, J. and Tabeling, P.,
(2006) 'PDMS-based microfluidics for proteomic analysis', The Analyst, Vol. 131,
No 10, pp. 1122-1128.
104 Mata, A., Fleischman, A.J. and Roy, S., (2005) 'Characterization of
Polydimethylsiloxane (PDMS) Properties for Biomedical Micro/Nanosystems',
Biomed. Microdev., Vol. 7, No pp. 281-293.
105 Siegel, A.C., Bruzewicz, D.A., Weibel, D.B. and Whitesides, G.M., (2007)
'Microsolidics: Fabrication of Three-Dimensional Metallic Microstructures in
Poly(dimethylsiloxane)', Adv. Mater., Vol. 19, No 5, pp. 727-733.
106 Xia, Y., Rogers, J.A., Paul, K.E. and Whitesides, G.M., (1999) 'Unconventional
Methods for Fabricating and Patterning Nanostructures', Chem. Rev., Vol. 99, No 7,
pp. 1823-1848.
107 Efimenko, K., Wallace, W.E. and Genzer, J., (2002) 'Surface Modification of
Sylgard-184 Poly(dimethyl siloxane) Networks by Ultraviolet and Ultraviolet/Ozone
Treatment', J. Colloid Interface Sci., Vol. 254, No 2, pp. 306-315.
108 Uyama, Y., Kato, K. and Ikada, Y. (1998) Surface Modification of Polymers by
Grafting, in Grafting/Characterization Techniques/Kinetic Modeling. Springer:
Berlin.
109 Heyries, K.A., Marquette, C.A. and Blum, L.J., (2007) 'Straightforward Protein
Immobilization on Sylgard 184 PDMS Microarray Surface', Langmuir, Vol. 23, No
8, pp. 4523-4527.
110 Heyries, K.A., Blum, L.J. and Marquette, C.A., (2008) 'Direct
Poly(dimethylsiloxane) Surface Functionalization with Vinyl Modified DNA',
Chem. Mater., Vol. 20, No 4, pp. 1251-1253.