22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Aerosol-assisted atmospheric-pressure plasma deposition: a versatile tool for the
one-step production of protein-embedded coatings
G. Camporeale1, G. Dilecce2, C. Loporto1, F. Palumbo2, E. Sardella2 and P. Favia1,2
1
Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via Orabona 4, IT-70124 Bari, Italy
2
Istituto di Metodologie Inorganiche e dei Plasmi CNR Bari, Via Orabona 4, IT-70124 Bari, Italy
Abstract: In the present work we report on our last outcomes on an atomizer-assisted
atmospheric-pressure plasma process for the production of coatings obtained by the
co-deposition of an organic precursor and water or water solution containing a bioactive
molecule, lysozyme and vancomycin
Keywords: aerosol-assisted plasma, atmospheric-pressure plasma, bio-composite coating
1. Introduction
Bio-composite coatings can be defined as interlayers
made of at least two components: an organic/inorganic
synthetic matrix, well adherent to substrates and working
as supporting network, and biological compounds (such
as proteins, nucleic acids, lipids, and even cells, viruses or
their fractions) embedded, conjugated or mixed to the
matrix. They are normally employed to improve the
biological
activity
(antibacterial,
cell-adhesive,
biomolecule-sensing, etc.) of the material they are
deposited on, without altering its bulk properties.
This kind of bio-composite films can be exploited in a
wide range of technological applications including
biosensors, cell growth enhancing coatings for tissue
engineering, antibacterial films for food packaging [1-3].
For this reason many techniques, such as drop-casting, dip
coating and painting [4-6], layer-by-layer deposition of
polyelectrolytes
[7,8],
electrochemically
induced
deposition [9], sol-gel [10], many wet chemical reactions
[11-13], have been developed in order to synthesize biocomposite coatings. Unfortunately, all of them contain
some drawbacks (e.g., large use of solvents and reactants,
long tedious multi-step procedures, need for sample
pre-treatments, etc.) that can be overcome using
plasma-phase reactions. So far, two main strategies based
on reactions in plasma phase have been set up. One
consists in a two-step process, in which substrates are
firstly functionalized by either atmospheric- or
low-pressure plasmas, then grafted with biomolecules in a
wet chemistry step [14, 15].
Recently many efforts have been devoted to set up onestep atmospheric-pressure plasma-based processes leading
to embed biomolecules in polymeric thin films. This
strategy can be carried out by coupling Dielectric Barrier
Discharge (DBD) reactors with atomizing systems
spraying nano-drops of biomolecule solution directly in
the plasma chamber.
This group of research has already demonstrated the
possibility to exploit this strategy to successfully include
active lysozyme, an antibacterial protein, in plasmadeposited polymeric coating bulk structures, using
P-III-6-8
ethylene as precursor for the synthesis of the matrix. The
one-step procedure did not influence the functionality of
the enzyme and allowed its tuneable release in water. This
could pave the way for the application of aerosol-assisted
atmospheric-pressure plasma deposition of bio-composite
coatings to the dry manufacturing of drug carrier systems
with modulated delivery behaviour.
At the best of our knowledge, in the present work we
report the first effort in scientific literature to create biocomposite organosilicon coatings embedded with
bioactive proteins by means of aerosol-assisted
atmospheric-pressure plasma deposition. In particular, the
influence of the addition of sprayed water and lysozyme,
an antibacterial enzyme, watery solution on the chemical
composition of coatings deposited from HMDSO in a
DBD has been analysed, as well as the behaviour in
aqueous environment of the so produced films.
Furthermore results are also reported concerning a similar
process obtained when the matrix precursor is ethylene
and the bioactive molecule is Vancomycin, a common
antibiotic, with a molecular weight 10 times lower than
lysozyme.
2. Materials and Methods
The DBD reactor is schematically represented in Fig. 1.
It consists of two parallel plate silver electrodes, 5 mm
apart, both covered by thick alumina sheets. Helium
(carrier gas, 99.999% Air Liquide) was fed through
electronic mass flow controllers (MKS Instruments). For
the lysozyme containing composite, HMDSO was
introduced in the feed bubbling He at a flow rate of
25 sccm in a stainless steel reservoir kept at constant
temperature, 25 °C, by means of a thermostatic bath.
Water aerosol was added to the feed with an atomizer
(mod. 3076 TSI) operated with He at 3 and 5 slm flow
rate, and water was replaced with a lysozyme solution for
the bioactive composite coatings.
For the Vancomycin containg films, C 2 H 4 was used as
the precursor of the matrix at the flow rate of 5 to
10 sccm. In this case water was replaced with a
vancomycin solution. Before each deposition process, He
1
broad band at 3755-3100 cm-1 and the Si-OH stretching
feature (910 cm-1), instead, appear when atomized water
is injected in plasma and become more intense when the
flow of He through the aerosol generator is increased
from 3 to 5 slm.
Fig. 1.
Schematic diagram of aerosol-assisted
atmospheric-pressure DBD deposition system.
was added to the reactor chamber for 10 min for
conditioning and purging. The discharge was ignited
using an AC power supply consisting of a function
generator (TG1010A, Thurlby Thander Instruments), an
amplifier (Industrial Test Equipment Powertron 1000A)
and a high voltage transformer (Amp Line). The
electrical properties of the plasma were investigated for
determining the voltage and the current delivered to the
system with a high voltage (P6015A, Tektronix) and a
resistance type current probe, both connected to an
oscilloscope (TDS 2014C, Tektronix). The average
power was obtained by multiplying the energy per voltage
cycle by the frequency; the energy per cycle was
calculated from the time integral of the current times the
voltage in one cycle. The applied voltage was kept at
6 kV pp at 4 kHz, corresponding to an average power
density value of 0.2 W cm-2.
Coatings were deposited on double side polished silicon
(100) shards (MIcroChemicals); their properties such as
wettability and chemical composition were evaluated with
a water contact angle goniometer equipped with a CCD
camera (CAM 2008, KSV Instruments) and a FT-IR
spectrometer (Vertex 70V Bruker), respectively. The
capability of lysozyme to leach out from the coatings was
verified by immersing samples in 2 mL of bi-distilled
water and by analysing the water after immersion with a
fluorescence spectrometer (Varian Cary Eclipse,
λ exc = 280 nm, λ em =350 nm).
3. Results and Discussion
3.1. Plasma deposition of lysozyme/HMDSO composite
coatings
The effects of water addition on the chemical
composition of plasma-polymerized HMDSO films have
been firstly investigated. Fig. 2 reports the normalized
FTIR spectra of films deposited without water and with
water atomization. For the first kind of films, the
following band assignments could be made: asymmetric
and symmetric C-H stretching (2965 and 2900 cm-1),
Si(CH 3 ) x asymmetric bending (1265 cm-1), Si-O-Si
asymmetric stretching (1090 and 1030 cm-1), Si(CH 3 ) 3
and Si(CH 3 ) 2 methyl rocking (840, 760 cm-1 and 800 cm1
, respectively). The features related to hydrocarboncontaining moieties are common to all the spectra, but
their relative intensity progressively decreases while the
water content in the system increases. The OH-related
2
Fig. 2. FTIR analysis of films deposited with no water
injection (a) and with 3 and 5 slm (b and c, respectively)
atomizer flow rates.
All the data seem to reveal the oxidative behaviour of
water-derived radicals (e.g. OH, and O and H atoms).
Their interaction with methyl group of the precursor is
supposed to lead to the formation of highly volatile
compounds (such as methane, carbon oxides, silane,
methylsilanes, etc.), that are removed from the reaction
environment through the aspiration system.
According to FTIR data, during the deposition some
hydrophobic methyl groups are substituted by hydroxyl
moieties, more prone to form hydrogen bonds with water;
this could explain the decrease of water contact angle of
the coatings deposited from a HMDSO/H 2 O mixture
compared to the ones produced without water addition
(Table 1). Nevertheless, it must be underlined that the
coatings do not really become hydrophilic even when
massive quantities of water are injected in the plasma.
Table 1. Static, advancing and receding water contact
angles of coatings produced from HMDSO/H 2 O
precursors at 4 kHz and 6 kV pp .
He flow rate
through atomizer
stWCA
advWCA
recWCA
5 slm
93 ± 1
99 ± 3
70 ± 3
3 slm
102 ± 2
106 ± 2
76 ± 4
0 slm
109 ± 1
112 ± 3
102 ± 4
P-III-6-8
Fig. 3 represents the normalized FTIR spectra of the
films deposited under various atomizer flow rate
conditions when water was substituted with a watery
solution of lysozyme (8 mg/mL), together with the
absorption spectrum of pure Lyz. All the deposited
coatings exhibit an organosilicon backbone, revealed by
the absorption bands relative to CH 3 asymmetric and
symmetric stretching (2965 and 2900 cm-1), Si(CH 3 ) x
asymmetric bending (1265 cm-1), Si-O-Si asymmetric
stretching (1090 and 1030 cm-1), Si(CH 3 ) 3 methyl
rocking (840 and 760 cm-1) and Si(CH 3 ) 2 methyl rocking
(800 cm-1). The addition of atomized lysozyme solution
in the reaction environment led to the appearance of new
bands in the spectra (C=O stretching at 1660 cm-1 and NH
bending at 1537 cm-1). Moreover, the broad band
between 3755 and 3100 cm-1 due to OH stretching (wide,
typically falling around 3400 cm-1) seems shifted towards
lower wavenumbers (from 3645 to 3055 cm-1, with a
maximum falling around 3310 cm-1). Such features are
compatible with the presence of amide bonds in the
spectra, that is consistent with the presence of the protein
(or its fragments) embedded in the structure of the
coating.
Fig. 3. FTIR analysis of casted Lyz (a), films deposited at
3 and 5 slm atomizer flow rates of water aerosol (b and d)
and of 8 mg/mL solution of Lyz (c and e).
In order to study the behaviour of these lysozymeembedded composite coatings in an aqueous environment,
samples were immersed in distilled water for 1 day and
the coatings were analysed by means of FTIR absorption
spectroscopy before and after immersion, while the
extracted solution was examined with a fluorescence
emission spectrophotometer. Almost no changes neither
in FTIR composition nor in the relative intensity of the
features could be detected after immersion in the coatings
(Fig. 4). In particular, the FTIR features characteristic of
the presence of proteins in the films underwent no
intensity variation before and after immersion. This
observation, together with the lack of relevant signals
detected
from
the
fluorescence
emission
P-III-6-8
spectrophotometric analyses of the immersion solution,
led to hypothesize that no lysozyme release from the
coating occurred.
Fig. 4. FTIR analysis of films before (a and c) and after
(d and d) water immersion. Films plasma-deposited at
5 slm atomizer flow rate without any atomizer flow (a and
b) and fed with an 8 mg/mL Lyz solution.
3.2. Plasma deposition of vancomycin/C 2 H 4 composite
coatings
The deposition process of the vancomycin containing
composite films has been optimized, working in
continuous and pulsed mode. In Fig. 5, the FTIR spectra
of vancomycin containing coatings deposited in
continuous and pulsed mode at the same peak power are
reported together with that of the pure antibiotic. It can
be observed that in continuous mode the presence of
bands that can be attribute to vancomycin is negligible.
However when decreasing the flowrate of ethylene from
10 to 5 sccm (Fig. 5 C and D) the spectrum appearance
pass from that typical of an hydrocarbon plasma deposited
coating to one having some features that can be attributed
to vancomycin (especially the broadening of the OH/NH
region at 3300 cm-1).
More interestingly, when passing from the continuous
mode to the pulse one, an important contribution of the
fingerprints bands of vancomycin appears (Fig. 5B).
This can be surely due to the decreased effect of the
power onto the fragmentation of the antibiotic molecule.
In particular it can be expected that during the off time
vancomycin can be adsorbed on the growing coating
without important damage of its structure.
It is important to stress the difference between the
lysozyme and vancomycin cases, in the deposition of such
bio-composite coatings. In fact while lysozyme can be
trapped in similar plasma polymerized films even in
continuous plasma, vancomycin required pulsing of the
discharge. This could be likely ascribed to the lower
molecular weight of vancomycin [17].
3
Absorbance
A
B
C
D
4000
3600
3200
2800
2400
1800 1600 1400 1200 1000
800
Wavenumber (cm-1)
Fig. 5. FTIR analysis of casted vancomycin (A), films
deposited in pulsed mode (B, 10 ms:100 ms t ON :t OFF , 5
sccm of C 2 H 4 ), continuous mode at 5 sccm of C 2 H 4 (C)
and 10 sccm of C 2 H 4 (D). Power density of 0.75 W/cm2,
He flow rate in the atomizer of 5 slm, 10 mg/mL solution
of vancomycin.
[9] P. Lisboa, et al. Micro Nanosyst., 3, 83 (2011)
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(2008)
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[16] P. Heyse, et al. Plasma Process. Polymers, 8, 965
(2011)
[17] P. Favia, G. Camporeale, Y.-W. Yang, E. Sardella,
G. Dilecce and F. Palumbo. in: 4th Int. Conf. on
Plasma Surface Engineering. (September 15-19)
(2014)
4. Conclusions
An aerosol-assisted atmospheric-pressure plasma
deposition process has been used to deposit
HMDSO/lysozyme and vancomycin/CHx coatings. It has
been found that Lyz containing coatings were not able to
release the entrapped protein into water, probably because
of their marked hydrophobic behaviour.
However
attempts in increasing the hydrophilic character of the
surface and in turn the releasing attitude are ongoing.
On the other hand pulsed mode conditions have been
successfully found for the deposition of vancomycin
composite films. Wide possibilities of optimizing this
process for further development in drug delivery systems
can be envisioned.
5. Acknowledgements
Mr Savino Cosmai (IMIP-CNR), Mr Danilo Benedetti
(University of Bari) are acknowledged for their valuable
technical assistance.
The projects LIPP (Rete di
Laboratorio 51, Regione Puglia) and SISTEMA (PON
MIUR) are gratefully acknowledged for funding and
supporting this research.
6. References
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