Hydration of Hydrophobic/Hydrophilic Plasma Polymer Films

Affecting Protein Adsorption

D. Hegemann1, N.E. Blanchard1, N. Hocquard1,2 and M. Heuberger1
1
Empa, Swiss Federal Laboratories for Materials Science and Technology, St.Gallen, Switzerland
2
ECPM, Ecole Européenne de Chimie, Polymères et Matériaux, Strasbourg, France

Abstract: The control of protein adsorption – the first step at material surfaces that are
exposed to a biological medium – is crucial for all biomaterials. In addition to the common
surface-related interaction forces (surface energy, charging, hydrogen bonding, molecular
forces), a plasma polymer film comprising a hydrophobic/hydrophilic vertical chemical
gradient nanostructure was found to affect the interaction forces due to water penetrating its
subsurface. As a result, reduced protein adsorption was observed for BSA.
Keywords: plasma polymerization, subsurface effect, water structuring, protein adsorption

1. Introduction
The common definition of “surface” includes surface
atoms and molecules, practically extending at the most
some three layers – typically one nanometer. This
definition is justified by the fact that many surface
properties related to symmetry breaking, such as
chemistry, wettability or charge density are determined by
the top most surface layer. The common understanding is
that this thin surface region also determines how
molecules adsorb onto it.
Far less explored are effects due to interactions with
deeper subsurface layers, i.e. the region extending over
several nanometers underneath the “surface”. This
subsurface region, however, might significantly
contribute to molecular adsorption via long-range (i.e.
few nm) interaction forces; mainly interactions with fixed
dipoles, water structuring and Van der Waals interactions
[1]. A key factor to make use of these interaction forces
thus lies in the hydration of the subsurface region.
Therefore, stable plasma polymer films made of siloxanes
were designed that contain a hydrophilic nanoporous base
layer terminated by a hydrophobic top coating, nominally
2-8 nm thick (Fig. 1) [2]. As a model molecule, bovine
serum albumin (BSA) was selected and its adsorption was
studied on gradient coatings as well as reference coatings
immersed in water or phosphate buffered saline (PBS).
Fig. 1. Schematic drawing of the examined
hydrophobic/hydrophilic plasma polymer films with
vertical chemical gradient nanostructure.
2. Experimental
Hexamethyldisiloxane (HMDSO), (CH3)3Si-O-
Si(CH3)3, was used as starting molecule (monomer) with
a gas flow rate of 4 sccm with or without addition of
oxygen (40 sccm) in the discharge. Argon was used as
carrier gas (20 sccm). The RF plasma (13.56 MHz) was
capacitively generated by a steel electrode (21 x 70 cm2)
mounted inside a vessel with a volume of 40 l [2]. The
pressure was fixed at 7 Pa, while power input was 50 W
for HMDSO/Ar discharge without oxygen and 100 W for
oxygen addition [3]. Beside the deposition of reference
coatings using such fixed deposition condition (50 nm
thick), vertical chemical gradient films were deposited by
using the experimental conditions with O2 addition as
base layer (50 nm) and the conditions without O2 as top
layer with varying thickness of 2, 4, and 8 nm. All films
were smooth with a rms roughness of about 0.3 nm (1 x 1
µm2) as measured by AFM. Protein adsorption was
examined using a white light interferometric method
called Transmission Interferometric Adsorption Sensor
(TInAS) on plasma-coated sensors measuring the
(average) thickness of an adsorbate [2,4]. Bovine serum
albumin (BSA) served as model protein which was added
at 5 mg l-1 in distilled water or phosphate buffered saline
(PBS).
3. Results and Discussion
Using plasma polymerization, either hydrophobic or
hydrophilic plasma polymer films (PPFs) can be
deposited from HMDSO discharges mainly depending on
the admixture of an oxidizing agent such as oxygen.
While highly oxidized, dense quartz-like films can be
achieved as used for barrier coatings, the PPF properties
can also be tuned by the residual hydrocarbon content as
well as the densification of the films within the non-
equilibrium plasma environment. Nanoporous plasma
polymers (ppSiOx) can thus be observed, where water
molecules are able to penetrate this moderately
hydrophilic nanostructure [5]. Without oxygen admixture
PDMS-like plasma polymers (ppHMDSO) can be
deposited which are hydrophobic due to their high
number of residual CH3 groups bound to a stable,
branched Si-O backbone. Both PPFs were used as
reference coatings (50 nm thick).
Furthermore, the 50 nm ppSiOx film was used as base
layer coated by a 2, 4 or 8 nm thick adlayer of
ppHMDSO. Interphase formation in plasma polymer
deposition yields the formation of vertical chemical
gradient structures, while the ppHMDSO layer entirely
covers the base layer as indicated by water contact angle
(WCA) and topographical (roughness) measurements
(Fig. 2).

Fig. 3. Adsorbed BSA thickness for the studied siloxane

films as measured by TInAS with water. Note that 1 nm
corresponds to a deposited mass of ~140 ng cm-2 for BSA.

To prove the hypothesis that hydration of the vertical

Fig. 2. Water contact angles measured on reference
coatings (ppSiOx, ppHMDSO) and vertical gradients
(with nominally 2, 4, and 8 nm hydrophobic adlayer).
Water penetration into such PPFs was investigated by
neutron reflectometry (using D2O) showing that water
readily penetrates ppSiOx films [5]. Moreover, water was
also found to slowly diffuse into ppHMDSO films, up to a
depth of 10 nm over a period of 4 hrs immersion.
For a typical adsorption measurement with BSA, the
siloxane PPFs were deposited on optical sensors and pre-
immersed in water or PBS for 10 hrs. The optical method
TInAS allowed to follow adsorption kinetics of BSA [2].
A stable base line was observed for all cases proving the
stability of the PPFs in aqueous environments.
Interestingly, the protein adsorption on all vertical
gradient films was found to be reduced – even below the
adsorption on ppHMDSO (Fig. 3). Note that also BSA
adsorption on ppSiOx is noticeably reduced compared to
quartz surfaces that typically show full coverage with
BSA of about 4 nm thickness. The same values of
adsorbed BSA have also been obtained with PBS instead
of water showing a negligible influence of ions (for the
used concentration).
gradient nanostructures is a crucial factor affecting the
observed protein adsorption, a 4 nm vertical gradient film
was prepared without pre-immersion, i.e. starting the BSA
addition immediately after immersion within the TInAS
device. Different to the pre-immersed samples, the “dry”
gradient PPF revealed the same adsorption as on
ppHMDSO (Fig. 4). Hence, BSA adsorption is mainly
governed by surface energy on siloxane films as long as
subsurface effects are absent. The immersion time, on the
other hand, enables water to penetrate through the
hydrophobic top coating reaching the hydrophilic base
layer, where water can get trapped in the subsurface
forming water structuring throughout the vertical
gradient. It can be assumed that the density of water
molecules at the surface, which is low for hydrophobic
surfaces, is thus increased by hydrogen bonds between
water molecules at the interface enabling a different
conformation of adsorbed proteins [6,7]. Moreover,
hydration of the subsurface might result in fixed dipoles
and a change in polarity affecting the long-range
interaction forces [8].

Fig. 4. Adsorption curves for pre-immersed and dry

vertical gradient films compared to ppHMDSO.
 Another experimental series using the 4 nm thick
gradient layers has been conducted, this time in PBS, by
introducing a short drying cycle for 3-15 min, i.e. PBS
was removed, directly before re-immersion and BSA
addition (Fig. 5).
Fig. 5. Adsorption curves for pre-immersed vertical
gradient films. PBS was removed for certain periods
shortly before the addition of BSA.
exerted by the subsurface, was revealed in addition to
known surface-related forces, which is an important
finding for the biomedical and related fields.
5. References
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Shell, J. Israelachvili, Faraday Discuss., 146, 299 (2010)
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40, 7245 (2007)
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By examining the effect of the different drying cycles,

it was found that already a 3 min drying period before re-
immersion resulted in enhanced BSA adsorption
compared to the permanently immersed sample. Longer
drying periods yielded a further increase in BSA
adsorption reaching the properties of ppHMDSO films for
10 min or longer periods.
It can thus be assumed that the water structuring
through the hydrophobic adlayer is delicate and can be
interrupted by drying within several minutes.
Interestingly, the BSA adsorption remains stable
(saturated) although a re-hydration might be expected.
Most likely, BSA proteins become denatured at (non-
hydrated) hydrophobic surfaces being a non-reversible
process [9]. The intermediate behavior for 3 and 5 min
drying periods would thus indicate partly denaturation. It
might further be inferred that less denaturation is
observed on the hydrated vertical gradient structures.
Hydration of hydrophobic/hydrophilic plasma polymer
films can thus be further exploited to control protein
adsorption and activity.

4. Conclusion
Plasma polymer films comprising a hydrophobic/
hydrophilic vertical nanostructure based on siloxane film
chemistry using HMDSO discharges were investigated
which are exceptionally stable in aqueous environments.
Protein adsorption with BSA can thus be observed using a
sensitive, optical method, TInAS, for dry and hydrated
gradient PPFs. Water penetration into the gradient
structure was found to affect protein adsorption as long as
water structuring, presumably by hydrogen bonding,
between the trapped water in the subsurface and the
aqueous environment persists. Hence, an additional factor
to control protein adsorption, namely interaction forces