Comparison of localized treatment effectiveness on biocompatible glass with
different atmospheric pressure plasma sources
Marco Boselli2, V. Colombo1,2, E. Ghedini1,2, M. Gherardi1, R. Laurita1,
A. Liguori1, F. Marani1, P. Sanibondi1 and A. Stancampiano1
Alma Mater Studiorum – Università di Bologna
1
Department of industrial engineering (DIN)
2
Industrial Research Center for Advanced Mechanics and Materials (C.I.R.I.-M.A.M.)
Via Saragozza 8-10, 40123 Bologna, Italy
Abstract: A comparison of localized treatment effectiveness on biocompatible glass substrate
with four non-thermal atmospheric plasma sources is presented. The aim is to identify the best
plasma source and operating conditions to create definite patterns on the substrate.
Investigated sources: Dual Gas Plasma Jet, DBD, Micropen Plasma Jet and kINPen 10. Local
modification of the substrate is evaluated with water contact angle (WCA) measurements.
Keywords: non-thermal atmospheric pressure plasma, localized plasma treatment, WCA,
biocompatible, glass, cell culture coverslip
1. Introduction
In recent years, increasing efforts have been dedicated to
atmospheric pressure non-thermal plasma technology for
the modification of materials surface characteristics. The
many and wide varieties of non-thermal plasma
applications for material modification are due to the
electric fields and the mixture of ions, electrons, photons,
neutral and excited molecules that co-exist in the ionized
gas plasma[1]. An important research topic in the field of
material treatment regards the modification of biomaterials
to promote cell adhesion, growth and proliferation [2].
Non-thermal plasma modification is gaining popularity
in the medical field because of its potential of changing the
chemical composition and properties of biomaterials (such
as wettability and chemical inertness) introducing desired
functional groups or chains onto the material surface able
to improve the material biocompatibility [3]. Moreover
another interesting characteristic of plasma technology is
its environmental compatibility: plasma processes don’t
require any chemical compound unlike conventional
chemical technologies [4].
Thought low pressure non-thermal plasma is already an
established technology in the field of material modification
[5], atmospheric pressure plasmas are rising interest
because of their easy handling, effectiveness and low
operational costs. Moreover, the possibility of locally
surface modification and the creation of patterns, having
different chemical composition and surface characteristics
from the remaining area of the substrate, would be of
great interest in many different fields, from tissue
engineering to biosensors production.
In this work non-thermal plasma modification at
atmospheric pressure of borosilicate glass cell-culture
coverslips, with a particular focus on the localization of
the plasma treatment, has been performed.
Four non-equilibrium atmospheric pressure plasma
sources have been tested: a Dual Gas Plasma Jet, a
Dielectric Barrier Discharge (DBD) and a Micropen
Plasma Jet, all three powered by a high voltage pulse
nanopulsed generator, and a commercial plasma source
(kINPen 10, Neoplas Tools GmbH). Surface wettability
before and after plasma treatment has been studied using
WCA measurement.
2. Materials and methods
Substrate
The tested biocompatible substrate is borosilicate glass
cell-culture coverslip with a surface area of 24x24 mm,
and a thickness of 0.13-0.16 mm.
Non-thermal atmospheric pressure plasma sources
The Dual Gas Plasma source is a single electrode
plasma jet developed by the Authors and already presented
in details in a previous article [6]. Two separate gas inlets
are employed to control the composition of the plasma and
the production of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) as evaluated in [7]; the
primary gas is usually Ar or He, while the secondary gas is
generally O2, N2 or Air.
The DBD is one of the most investigated non-thermal
plasma sources and it is usually found in three
configurations: the standard DBD, in which the discharge
is ignited between a high voltage electrode and a (usually
grounded) counter-electrode; the floating electrode DBD
(FE-DBD), in which the material to be treated plays the
role of the counter-electrode; the indirect DBD, in which
the material to be treated is only exposed to the effluent of
the DBD discharge. Plasma source used in the present
work consists of a cylindrical copper electrode (diameter
of 26.2 mm), a dielectric plastic layer (thickness 1.5 mm)
surrounding the electrode body along its length and a thin
quartz disk (diameter 30 mm, thickness 0.5 mm) covering
the electrode plane surface facing the plasma forming
region.
The Micropen Plasma Jet is an innovative non-thermal
plasma source, projected and realized by the Authors, able
to generate a plasma jet protruding from a needlelike
output having a diameter of 600 µm. This plasma source
can operate ionizing different gas such as He, Ar and air.
Thanks to the micrometric size of the output, the source
can be successfully used for the creation of defined
patterns on the substrate, promoting alterations of chemical
composition and surface characteristics only in the
localized regions directly exposed to the plasma jet.
In this work all three non-thermal plasma sources
described above have been powered by a nanosecond pulse
generator having a peak voltage (PV) between 7 and 20 kV
into a 100-200 load impedance, a pulse repetition
frequency (PRF) between 50 and 1000 Hz, a pulse width
(50%) of 12 ns and a rise time of 3 ns. Plasma sources can
be also driven by an high voltage amplifier connected to a
function generator operating with several waveforms such
as sinusoidal, triangular, square and sawthooth.
The APPJ kINPen 10 is a commercial plasma source
projected and realized by the Leibniz Institute for Plasma
Science Technology (INP Greifswald).
The whole
system consists of a hand-held unit, a DC power supply
and a gas supply unit that works with all rare gas, but also
with air and N2. Plasma is ignited at the tip of a centered
electrode inside the capillary and blown out by the gas
flow to form the plasma jet [8] [9].
Measurement of water contact angle
The modification of chemical composition and surface
properties of the borosilicate glass cell-culture coverslip
surface is investigated by means of advancing and
receding WCA measurements. The instrument used for
the analysis is the Kruss Drop Shape Analysis System
DSA 30. Two different methods were experimented to
evaluate the variation of surface wettability: sessile drop
method and tilting plate method.
Sessile Drop Method (SD): a drop of distilled water is
at first deposited on the sample surface by a micrometric
syringe and its volume is increased forcing the drop to
advance on the sample, until a constant contact angle is
observed, representing the advancing contact angle. Then
the drop is progressively shrunk until a drop radius
decrease is obtained [10].
For the measurements in the cases of samples treated by
Dual Gas Plasma Jet, DBD and kINPen the drop of
distilled water at first deposited had a volume of 4 µl,
while in the cases of samples treated by Micropen Plasma
Jet the drop volume is of 1 µl.
Tilting Plate Method (TP): a drop of distilled water is
released by a micrometric syringe on a tilted plate having
an inclination of 6° to evaluate the maximum advancing
water contact angle when the drop is free to move. The
advancing WCA is the angle of drop profile in the
direction of motion, while the receding angle is that
opposite to the direction of drop motion. For the
measurements in the cases of samples treated by Dual Gas
Plasma Jet, DBD and kINPen the drop is of 4 µl, while in
the cases of samples treated by Micropen Plasma Jet the
volume realized is of 1 µl.
To analyze the variation of surface wettability, several
measurements havebeen carried out both on the untreated
and the plasma treated substrate. Moreover, to verify the
localization of the plasma treatment, a high number of
WCA measurements have been effectuated on different
points of the same substrate to evaluate the different
values obtained in the non-exposed and directly exposed
regions.
3. Results
The advancing water contact angle values for the
unmodified borosilicate glass coverslip obtained with
sessile drop and tilting plate methods are 56°±3° and
42°±9° respectively, while the receding values with
sessile drop and tilting plate methods are 53°±3° and
31°±3° respectively.
To evaluate the localization of plasma treatment for
Dual Gas Plasma Jet, DBD and kINPen 10 cases four
regions (quadrants) have been identified onto each slide
and the plasma discharge has been oriented only towards
one of these. In Fig. 1 a schematic representation of the
slide division is represented identifying with P the
quadrant exposed to the plasma and with U the other
quadrants not directly subjected to the discharge.
Fig.1 Quadrants. P: exposed to the plasma, U not
directly exposed to the plasma.
Dual Gas Plasma Jet
Plasma with the Dual Gas Plasma Jet has been
generated using 2 slpm of Ar as primary gas without
injection of secondary gas. Peak voltage has been fixed to
34.8 kV, pulse repetition frequency to 1000 Hz, treatment
time has been varied from 10 s to 20 s and the gap
between plasma source and substrate from 15 mm to 25
mm. In Table 1 advancing and receding WCA have been
presented for different treatment time and gap between
plasma source outlet and substrate. Peak voltage has been
fixed to 34.8 kV, pulse repetition frequency to 1000 Hz
and 2 slpm of Ar has been used as mass flow rate.
Table 1. Dual Gas Plasma Jet: advancing (θa) and
receding WCA (θr) with SD and TP methods
Time
[s]
Gap
[mm]
20
25
30
25
20
15
Quad
rant
P
U
P
U
P
U
SD
θa[°]
17±3
42±5
9±2
60±3
8±2
52±1
SD
θr[°]
15±2
36±2
10±2
56±4
7±2
53±2
TP
θa[°]
8±1
60±2
5±2
60±2
6±1
48±2
TP
θr[°]
6±1
60±2
4±2
50±1
4±2
45±2
Fixing the gap at 25 mm, a notable localization of the
plasma treatment is registered by both WCA measurement
methods and the wettability variation is found only in the
slide quadrant directly exposed to the plasma jet, without
relevant variation of surface properties in the other
regions. The increase of plasma treatment time from 20 s
to 30 s for the 25 mm gap causes an additional, but not
very significant, decrease of the water contact angles
values only in the quadrant directly exposed to the plasma
jet.
The reduction from 25 mm to 15 mm of the gap
between plasma jet outlet and glass slide for an exposure
time of 20 s doesn’t seem to cause a relevant alteration of
the localization of plasma treatment.
In the quadrant directly exposed to the jet, for a gap of
15 mm the advancing and the receding WCA measured by
tilting plate method have the same values of those
registered by the same method for a gap of 25 mm, while
the advancing and receding WCA measured by sessile
drop method are lower of about 8° than those measured
for a gap of 25 mm.
Dielectric Barrier Discharge (DBD)
Since the glass slide is smaller than the circular section
of the DBD high voltage electrode, treatment localization
has been realized employing a grounded counter electrode
metal layer placed below one of the quadrants. The
quadrant below which counter electrode is placed has
been interested by the discharge while the others haven’t
been directly subjected to the plasma.
To obtain a diffuse plasma discharge, peak voltage has
been set to 24.4 kV, pulse repetition frequency to 384 Hz,
inter-electrode gap to 1 mm and treatment time to 10 s.
Both WCA measurement methods have noticed a drastic
increase of slide wettability in the quadrant interested by
the plasma discharge, while in the others no great
variations have been observed comparing the sample with
the pristine substrate.
In Table 2 the advancing and receding WCA values
obtained with sessile drop and tilting plate methods in the
quadrant (P) interested by the plasma discharge and in the
other quadrants (U) are presented.
Table 2. DBD: advancing WCA (θa) and receding WCA
(θr) with SD and TP methods
Quadrant
P
U
SD
θa[°]
11±1
63±3
SD
θr[°]
11±1
61±4
TP
θa[°]
15±1
58±2
TP
θr[°]
5±1
55±1
Micropen
Micropen plasma source has been investigated to
evaluate the feasibility of using the source to create a
micrometric highly hydrophilic pattern on the slide
without altering the surface properties of the untreated
area.
To verify the localization of the plasma treatment the
microjet has been moved along a defined stripe on the
substrate. The width of treated pattern has been evaluated
by means of several WCA measurements along the
pattern and in the adjoining regions. The area of the slide
exposed to the plasma has been estimated to be smaller of
1 mm in width. Water contact angle drastically decreases
in this region after only 10 s of treatment while in the
surrounding area no relevant wettability changes are
observable, as demonstrated by both WCA measurement
methods. The pattern subjected to the plasma jet and the
points in witch water contact angles have been measured
are schematically represented in Fig. 2.
The jet has been generated with a Ar flow injection or
in static air imposing a peak voltage of 30 kV, a pulse
repetition frequency of 500 Hz. Micropen Plasma Jet has
been moved to describe the definite pattern for 10 s at a
distance of 2 mm from the substrate.
In Table 3 the water contact angle values for different
operative conditions in the points represented in Fig. 2 are
proposed.
Fig.2 Pattern and points considered for the WCA
measurements
Table 3. Micropen Plasma Jet: advancing WCA (θa) and
receding WCA (θr) with SD and TP methods
SD
SD
TP
TP
Gas
Point
θa[°]
θr[°]
θa[°]
θr[°]
1
15±3
18±1
9±1
9±1
Ar 1
2
30±3
32±1
20±1
18±1
slpm
3
36±2
35±1
41±4
34±8
1
7±3
10±1
5±1
3±2
Static
2
14±1
15±1
7±1
7±1
air
3
51±3
50±1
32±1
38±1
Table 4. kINPen 10: advancing WCA (θa) and receding
WCA (θr) with SD and TP methods
Time
SD
SD
TP
TP
Quadrant
[s]
θa[°]
θr[°]
θa[°]
θr[°]
P
24±2
21±1
21±1
17±1
20
U
40±2
38±2
28±1
26±1
P
22±3
21±2
17±1
15±1
300
U
36±1
38±2
24±2
22±1
kINPen 10
With the kINPen 10, operated in Ar, there is no drastic
difference between the quadrant directly exposed to the
plasma and the remaining area. Power source of the
kINPen generator has been fixed to 3 W, Ar mass flow
rate to 3.2 slpm, gap between the plasma source outlet and
the slide to 25 mm.
For the kINPen 10 different treatment times have also
been tested increasing exposure time from 20 s to 300 s.
Sessile drop and tilting plate measurements allow to
deduce that the treatment time has not influence on the
increase of glass wettability, because no relevant
differences of the advancing and receding WCA values
can be noticed comparing the treated and untreated
regions of the slides exposed for 20 s with that of the
slides treated for 300 s.
The reduction of advancing and receding water contact
angle in the quadrant treated with the plasma is not
relevant and again the localization of plasma treatment is
not achieved because the decrease of the advancing and
receding WCA is not verified only in the quadrant directly
exposed to the plasma, but also in the other regions.
In Table 4 the effective values for different treatment
time of WCA in the quadrants (P) directly exposed to the
plasma and in the untreated quadrants (U) are presented.
4. Conclusions
Four non-thermal atmospheric plasma sources have
been tested to evaluate the possibility of locally modify
the surface properties of borosilicate glass cell-culture
coverslips. To evaluate the surface modification of the
substrate, sessile drop method and tilting plate method
have been used and the results obtained have been
compared. To verify the localization of plasma treatment,
for the cases of Dual Gas Plasma Jet, kINPen 10 and
DBD, the glass slide has been divided in four quadrants
and the plasma discharge has been directed towards only
one of these or generated in a restricted area. For the case
of the Micropen Plasma Jet, the localization of plasma
treatment has been investigated moving the plasma source
along a defined pattern on the substrate.
Advancing and receding WCA have been measured in
different points of treated and untreated regions.
With the Dual Gas Plasma Jet a great localization of
plasma treatment is registered by both methods in all
experimented configurations. The measurements obtained
using the tilting plate method don’t show any evident
correlation between the advancing and receding WCA
variation and the plasma treatment time or the gap
distance between source outlet and sample. The sessile
drop method results instead show a decrease of the WCA
of about 8° reducing the gap from 25 mm to 15 mm.
While with the Dual Gas Plasma Jet a localized
modification of the substrate wettability can be achieved,
with the kINPen 10 in comparison only a slight
modification is observed and not confined in the treated
region but on all the sample surface.
In the case of the DBD plasma source placing a
grounded counter electrode metal layer below one of the
quadrant a great increase of wettability is assured for very
short treatment time only in the region directly exposed to
the discharge while no significant variation is observed in
the remaining quadrants without underling metal substrate.
Results suggest the possibility to realize patterned
modification of large surfaces using DBD sources with
suitably shaped grounded counter electrodes.
For the Micropen Plasma Jet case, results highlight the
possible to achieve a millimetric defined hyperhydrophilic
pattern on the slide without relevant change in the
surrounding areas. For these results the Micropen can be
regarded as a promising tool to create highly defined
pattern having different characteristics from those of the
pristine substrate.
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