22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Plasma nanotextured polymer surfaces with tailored wetting properties and
superior performance in protein/cell immobilization
A. Tserepi1, E. Gogolides1, K. Tsougeni1, K. Ellinas1, A. Kanioura2, A. Bourkoula2, G. Kokkoris1, V. Constantoudis1,
P.S. Petrou2 and S. Kakambakos2
1
2
NCSR “Demokritos”, Institute of Nanoscience & Nanotechnology, GR-15310 Aghia Paraskevi, Attiki, Greece
NCSR “Demokritos”, Immunoassay/Immumosensors Lab, Institute of Nuclear and Radiological Sciences and
Technology, Energy and Safety, GR-15310 Aghia Paraskevi, Attiki, Greece
Abstract: In our work, plasma processing is employed to create micro- and/or
nanotextures on different polymeric surfaces. By appropriate tuning of plasma conditions,
either random or ordered hierarchical structures of high aspect ratio and surface area can be
created in a very repeatable manner. Such plasma treated polymeric surfaces have been
evaluated as substrates for controlling efficiently the wettability, biomolecule
immobilization, and cell adhesion, paving the way to a wide spectrum of applications.
Keywords: plasma nanotextured surfaces, superamphiphobicity, protein immobilization,
cell adhesion
1. Introduction
Surfaces with micro- and nanostructures acquire unique
properties, which render them appropriate for a variety of
applications.
For example, their resulting wetting
properties and control thereof at the extremes
(superhydrophobicity, superhydrophilicity) has acquired
an increased interest for applications such as
self-cleaning, anti-fogging, and anti-icing. In our work,
plasma processing has been employed as a versatile
technology to create micro- and/or nanostructures on a
variety of polymeric surfaces. By appropriate tuning of
plasma conditions, and in combination with other cost
effective lithographic processes (e.g., colloidal
lithography), both random and ordered hierarchical
structures of high surface area can be created on polymers
in a very repeatable manner [1-5]. Such micro- and
nanotextured surfaces combined with deposition of
hydrophobic coatings can lead to a wide range of
wettabilities
from
superhydrophilicity
to
superhydrophobicity and superolephobicity [6-9].
Apart from changing the surface topography, plasma
processing alters also the surface chemistry and facilitates
biomolecule immobilization with unique advantages
[10, 11]. Compared to plain substrates as well as to
typical microarray substrates (i.e., functionalized glass
slides), the plasma treated surfaces offer enhanced protein
immobilization and thus increased detection sensitivity,
and high spot uniformity.
Immobilization of
biomolecules onto the plasma treated surfaces is
accomplished
mainly
by
physical
adsorption;
nevertheless, covalent binding through reaction with the
chemical moieties created on the surfaces through the
plasma treatment can be also employed [12]. The plasma
treated surfaces provide also unique properties as
substrates for controlling or investigating the cell
behavior [11, 13].
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Herein, we develop a summary of the work we
performed on plasma-based micro- and nanotexturing of
polymeric surfaces and their implementation in
wettability and biomolecule/cell adhesion control. We
show that plasma etching provides a unique and versatile
tool for tailoring the physicochemical properties of
polymeric surfaces, which in turn can be used in a wide
spectrum of applications such as self-cleaning, protein
microarrays, and cell cultures. For each of these
applications, we show that the unique properties of
plasma micro- and nanotextured surfaces can be further
exploited by their incorporation in selected areas into
microfluidic devices for flow control, and protein or cell
detection.
This short article is structured as follows: We first
provide general experimental details and physicochemical
characterization of the plasma micro and nanotextured
surfaces, as well as we discuss the mechanisms
responsible for the observed richness in surface
morphology. Second, we describe the resulting surface
wetting
properties
and
their
control
from
superhydrophilicity to amphiphobicity.
Third, we
demonstrate the unique advantages of plasma
nanotextured surfaces as superior substrates for
biomolecule immobilization and their implementation for
the creation of sensitive immunoassays. Fourth, we
explore the role of plasma-induced surface nanotexturing
in controlling cell adhesion and proliferation. Throughout
this presentation, we point out the ultimate goal of our
work, which is the “smart” incorporation of such surfaces
inside polymeric microfluidic channels so that the
benefits of plasma processing, not only as a fabrication
technology but also as a surface functionalization tool,
can be transferred to the revolutionary field of Lab-on-achip based bioanalytics.
1
2. Surface morphology; hierarchical random, quasiordered, or ordered micro- nanostructures.
Mechanisms of plasma nanotexturing of polymers
Plasma etching or sputtering of polymers was found
early-on to cause roughening of the polymeric surface.
We have shown that a short (1-3 min) plasma etching step
in a high density helicon plasma reactor leads to the
creation of high aspect ratio polymeric nanofilaments,
which grow several micrometres in height within a few
minutes etching [7, 14]. The process was demonstrated to
be generic for all polymers (Fig. 1), provided that the
processing gas implemented was appropriate for the
polymer being etched (SF 6 for Si-containing polymers
such as poly(dimethyl)siloxane (PDMS), or O 2 for
organic polymers such as poly(methyl)methacrylate
(PMMA), polystyrene PS, and cyclo-olefin copolymer
COP). Three general types of surface morphologies were
observed in our work; random, quasi-ordered, and ordered
hierarchical surfaces, and methods were developed for the
characterization of their complex morphologies.
(b)
(a)
(c)
(d)
Fig. 1.
Types of plasma-induced topographies on
polymeric surfaces; (a) quasi-ordered, (b) random, and
(c) ordered hierarchical. (d) Average height of surface
topographies on polymers (PEEK, PMMA, PDMS, PS) as
a function of time.
Extremely long (> 3 μm) polymeric filamental
structures were found not mechanically robust when
2
brought in contact with liquids, and in such cases,
nanofilament bundling and height reduction occurred as a
result of surface wetting-drying [15]. On the other hand,
under specific plasma conditions, small, quasi-ordered
nanostructures can be formed as well.
This was
demonstrated on oxygen plasma etched polymers in
mildly etching plasmas, for example at zero bias
conditions. These phenomena verify that plasmas can
also direct the formation of organized structures [5, 16].
The search for mechanically robust micro- and
nanotextured polymeric surfaces led to the need of
combining plasma etching with a precedent lithography
step to create sturdy microstructures before nanotexturing.
In our work, colloidal microsphere lithography was
proposed as a cost-effective lithographic method and the
obtained surfaces were shown to be scratch and
chemically resistant upon immersion to water and
hexadecane for periods of months [17].
It was assumed and was confirmed by chemical surface
analysis that nanotexturing is due to differential etching of
the surface in the presence of etching inhibitors coming
from the reactor walls and the prevailing anisotropic
etching conditions [7, 16]. Simulation of nanotexture
formation and growth was presented [18] and confirmed
that the etch inhibitor mechanism can also lead to
organized nanostructure formation on surfaces, in
addition to the formation of randomly rough surfaces.
3. Wetting control
Plasma processing has been shown to allow creation of
the whole spectrum of surface wetting regimes depending
on the plasma chemistry and conditions used.
Immediately after plasma processing in O 2 , micro
nanotextured surfaces become superhydrophilic, as a
result of plasma-induced roughness formation combined
with chemical modification [7]. In addition, surface
nanotexturing was proven useful to retard hydrophobic
recovery of polymeric surfaces, where desirable.
However in recent literature, a lot of attention has been
given to superhydrophobic surfaces and their fabrication
technologies.
Nanostructured surfaces turn into
superhydrophobic, after a subsequent fluorocarbon
plasma deposition [7, 14]. Such surfaces exhibit selfcleaning behaviour (Fig. 2), while under conditions
retaining their transparency and enhancing their antireflectivity [19]. Plasma technology is proven most well
suited for fabrication of superhydrophobic surfaces,
especially when such surfaces are incorporated in
Microsystems and labs on chip, as plasma is often the
technology used for their fabrication. In our work,
plasma nanotextured superhydrophobic surfaces are
incorporated in microchannels to form passive valves
[20].
Superoleophobicity is more difficult to achieve, since the
surface tension of oils and alkanes is low and thus
spreading of such liquids on the surface is easier.
Nevertheless, huge progress has also been achieved in the
design and fabrication of superoleophobic surfaces.
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Fig. 2. Plasma nanotextured, superhydrophobic,
self-cleaning surface, as a water drop rolls off and
removes red powder along its path.
Several works on the topic have shown that the structure
profile is crucial for achieving superoleophobicity. In our
work, colloidal lithography followed by plasma texturing
of the polymer and fluorocarbon plasma deposition or
grafting of stable perfluorinated monolayers was shown to
achieve superoleophobicity, due to the isotropic plasmainduced re-entrant curvature of the structures [17].
Extremely
repellant
polymeric
surfaces
were
demonstrated with static contact angles for water and oils
> 153° and hysteresis < 10° (Fig. 3). Furthermore, it was
proven that on such surfaces the observed
superamphiphobicity is stable, for droplets impinging at
extremely high pressures (> 36 atm for water and > 7 atm
for lower surface tension liquids), for the first time in the
literature [21].
Fig. 3. Liquid drops (water, hexadecane, soya oil) on
plasma nanotextured amphiphobic PMMA surfaces.
4. Protein immobilization control
Nanostructured surfaces offer increased surface area,
which if combined with appropriate surface chemistry
may greatly facilitate biomolecule immobilization. In our
work, a 5x increase in immobilized protein was observed
on (few min) plasma textured polymeric surfaces
compared to flat ones (Fig. 4) [22-23]. Such textured
polymeric surfaces were proposed by our group for the
creation of protein (e.g., antibody) or DNA microarrays
[24], offering an increased sensitivity by a factor of 100x
due to stronger signal (3x-10x), and better spot uniformity
[22-24], facilitating reliable immuno-analysis.
The incorporation of plasma-induced functional groups
may be critical for protein immobilization, and especially
its stability and the maintenance of the protein biological
activity. Plasmas are used to create functional groups such
as C=O, COOH, and OH, employing appropriate
processing gases prior to biomolecule immobilization.
We showed that direct (i.e., without linkers or further
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Fig. 4. Fluorescence images of b-BSA and RgG spots
deposited by a nanoplotter on (left) 20-min O 2 plasma
nanotextured PMMA surface and (right) untreated
PMMA
plate.
The
topography
of
the
plasma-nanotextured surface is shown in tilt and
magnification (middle).
surface functionalization) covalent immobilization of
protein molecules is possible on organic polymers after
short (1-5 min) plasma-induced surface nanotexturing. In
addition, stable in time chemical functionality is
demonstrated after a fast thermal annealing step of the
plasma nanotextured polymeric substrates, able to retain
the majority of the immobilized biomolecules, even at the
harshest washing conditions.
Furthermore, the
immobilized biomolecules retain their activity intact, as it
is proven by the sensitive detection of Salmonella
lipopolysaccharides after immobilization of the respective
specific antibody on plasma nanotextured surfaces [25].
5. Control on cell adhesion
Surface properties including wettability and topography
have been documented as critical factors that can affect
cell behavior. In particular polymeric surfaces intended
for cell growth need to be treated in order to become
hydrophilic for effective cell attachment. As plasma
nanotexturing is shown to control both wettability and
topography, it was also shown by our group that plasma
nanotextured substrates may be used for cell culturing
[26]. By controlling the plasma nanotexturing of a
polymeric surface along with the culturing time of cells,
the attachment and viability of cells is controlled,
providing substrates for cell cultures. For a specific cell
line (3T3 fibroblasts), we observed increased adhesion
and proliferation on nanostructured surfaces after 1 day of
culture (Fig. 5, top), while this trend was reversed after 3
days of culture.
Preferential attachment of cells is also shown on
superhydrophilic plasma nanotextured polymeric surfaces
with respect to their superhydrophobic counterparts
created after deposition of a Teflon-like coating [27], on
open or interior microchannel polymeric surfaces (Fig. 5,
bottom). Currently, this work advances towards selective
culturing and enrichment of specific type of cells from
mixtures.
6. Conclusion
Plasma etching is emerging as a nanofabrication tool
that can create hierarchical random, quasi-ordered, or
ordered structures when properly controlled or in
combination with cost-efficient lithographic processes.
3
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Fig. 5. (Top) Representative fluorescence images of
surface adherent 3T3 fibroblasts after 1-day culture on
untreated flat (left) and on oxygen-plasma nanotextured
PMMA surfaces (right). (Bottom) Fluorescence images
of HT1080 cells cultivated on untreated (circles) and
plasma-nanotextured surrounding PMMA surfaces (left)
and on superhydrophilic and superhydrophobic
microchannel surfaces (right). Cells were stained for
visualization, F-actin (green) and nucleus (blue).
The obtained rich surface morphology and its controlled
chemical functionality provide unique tools in a large
range of applications such as self-cleaning surfaces,
biomolecule microarrays, and cell cultures. Particularly
promising is the incorporation of such “smart” surfaces in
biomicrosystems and laboratories on chip for chemical
and biological analysis, a field of intense research (see for
example our recent review [28]).
7. Acknowledgments.
We acknowledge the EU and Hellenic GSRT projects
which have financed this work (Nanoplasma FP6 project,
ITN-Plasma Physics for Advanced Manufacturing FP7
project,
DESIREDROP
Thales
project,
and
PlasmaNanoFactory Excellence Research Project).
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