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edited by
Zdenko Machala
Comenius University
Bratislava, Slovakia
Karol Hensel
Comenius University
Bratislava, Slovakia
and
Yuri Akishev
SRC RF Triniti, Troitsk
Moscow Region, Russia
Published by Springer,
P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
www.springer.com
v
vi Preface
Bratislava, Slovakia
Contents
Preface .............................................................................................................. v
ix
x Contents
xv
xvi List of Corresponding Authors
1.1 Introduction
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 3
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_1, Springer Science+Business Media B.V. 2012
4 K.-D. Weltmann et al.
Typically, these devices are intended to be applied in contact with or even inside the
human body. Hence, stringent hygienic standards must be kept in order to avoid
device associated infections. Sterility, which means a state of being free from viable
microorganisms, is generally one of the key requirements in the preparation process
of a complex medical device. But also the sufficient decontamination from other
substances e.g. pyrogens must be considered. The most effective sterilization method
is still the use of hot steam with a temperature of 121C or 134C [1]. However, the
hot steam sterilization of catheters and endoscopes is restricted due to their imple-
mented heat sensitive materials. Furthermore, fever provoking bacterial endotoxins
(pyrogens) are not removed by this process.
Low temperature sterilization methods like the low temperature steam steriliza-
tion with formaldehyde (NTDF) and the use of microbicidal gases e.g. ethylene
oxide (ETO) or hydrogen peroxide are commercially applied to reprocess heat sen-
sitive devices nowadays. Despite the advantage of avoiding thermal damage of the
medical product these methods have different disadvantages. Most of the used
microbicidal gases are highly toxic and carcinogenic so that special requirements
are necessary to guarantee sterilization personnels safety. Furthermore, these gases
strongly penetrate the materials of the medical devices. This results in long outgas-
times until a sterilized device can be used again. Another disadvantage appears if
the low-temperature sterilization process uses alternating pressure techniques or
generally works in the low-pressure regime. In both cases the pressure sensitivity of
the medical device must be considered.
Because of difficult to predict irritation especially of polymeric materials as well
as very high safety requirements, gamma or electron-beam irradiation, respectively,
are also not practicable sterilization methods in several cases.
An alternative decontamination method for complex medical devices is the use
of atmospheric pressure plasmas [2], where a multiplicity of different antimicrobial
agents interacts with the biological contaminant. These agents are radicals and
chemical products e.g. atomic oxygen (O), hydroxyl (OH), reactive oxygen (ROS)
and reactive nitrogen species (RNS), high energy UV radiation, charged particles,
alternating electric fields, heat as well as physical and chemical etch processes.
Until now many investigations have been made to distinguish what plasma agent
exactly dominates the microbiological inactivation [35]. In fact, this is an impor-
tant but also sophisticated task since it depends sensitively on the plasma source and
the experimental conditions. However, most of the investigations show that the
combination of all plasma agents have much more effect compared to the efficacy
of one single component. But not only the pure antimicrobial effect makes atmo-
spheric pressure plasma interesting for the decontamination of complex medical
devices. Moreover, their ability to etch and degenerate dangerous bacterial endotox-
ins and their ability to penetrate small cavities opens up new fields of application in
the medical decontamination sector.
During the last decade great efforts have been made in inventing new plasma
sources and adapt them to the decontamination specific conditions. Three exam-
ples, on which this paper is focused on, are the atmospheric pressure plasma jet
(called APPJ according to [6]), the dielectric barrier discharge (DBD) and the
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 5
microwave driven discharge (MDD). In comparison with the APPJ and the DBD,
which are classified as non-thermal plasmas with moderate gas temperature (close
to room temperature) the MDD is indeed a low temperature but thermal plasma [7, 8].
Hence, its gas temperature is typically much higher than room temperature and can
exceed 5,000 K. However, using thermal plasmas in remote mode, where the
plasma itself does not reach the contaminated object, but the produced radiation,
radicals and chemical compounds, enables the treatment of even heat sensitive
devices.
Usually, for plasma-based techniques to inactivate and/or remove microorgan-
isms the term sterilization is used. However, a sterilization method has to meet
strict and well-defined requirements. Therefore, we prefer the use of the term
decontamination or bio-decontamination meaning a general inactivation or
removal of unwanted biological contaminants, especially microorganisms [9].
In this contribution, practicable realizations of atmospheric pressure plasma
sources for decontamination and their antimicrobial efficacy are presented. Since
catheters and endoscopes are prominent examples for the group of complex medical
devices only plasma sources that are basically adaptable to the specific device prop-
erties like geometry or heat sensitivity are considered here. The microbicidal efficacy
is determined either on commercially produced catheters or on polytetrafluoroethyl-
ene (PTFE) test tubes, which are adequate to the biopsy channels used in real
endoscopes.
In this study three different atmospheric pressure plasma sources are used. The
APPJ is a 27.12 MHz RF-driven source with a mean power of 20 W [8, 10, 11].
It consists of a nozzle made of ceramics with an inner diameter at the nozzle outlet
of about 7 mm. In the center of the nozzle a needle electrode is axial situated and
coupled with the RF-voltage; the grounded electrode is a ring shaped structure
directly at the outlet of the nozzle. Typical gas flow rates are in the range of up to
20 standard liters per minute (slm) of argon. In this configuration the filamentary
discharge is ignited inside the nozzle and the excited, diffuse shining gas leaves the
nozzle and can be used for surface treatment. These kinds of jets can be arranged in
different arrays. Here, the jets are mounted in a ring-like structure or a T-type nozzle
is used to treat medical devices e.g. catheters (Figs. 1.4 and 1.5).
To apply a dielectric barrier discharge (DBD) towards the inner surfaces of medi-
cal devices, two different setups were used. The first works with an inner electrode
as shown in Fig. 1.1. Here, the medical device is simulated by an alternate use of
PTFE tube (d = 2 mm) and metal tubes (d = 2 mm). This is a typical material combi-
nation e.g. to connect the biopsy channel with the control unit in endoscopes. To
ignite a discharge inside the tubes an inner electrode (d = 0.2 mm) is completely
covered with a dielectric (quartz glass, d = 1.2 mm) and introduced into the tubes.
The metal tubes work as grounded electrodes, the inner electrode is connected with
6 K.-D. Weltmann et al.
Fig. 1.1 Electrode arrangement to treat the inner surfaces of a combination of PTFE tubes and
metals with a DBD
Fig. 1.2 Modified PTFE tube with bifilar helix electrode configuration to ignite a DBD inside
the tube containing (1) powered electrode, (2) grounded electrode, (3) outer tube, (4) inner tube,
(5) discharge, (6) power supply, (7) electrical circuit for measuring the consumed power
up to 15 kV sinusoidal voltage with a frequency in the kHz range. The gas flow is
kept constant at 1 slm argon.
The second setup to generate a discharge inside a long flexible PTFE tube is
based on a bifilar helix electrode configuration as presented in Fig. 1.2.
The modified tube consists of an inner tube (4) and an outer tube (3) concentrically
aligned. Intermediate, a powered (1) and a grounded (2) electrode are arranged equidis-
tant twisted around the inner tube. The distance between the electrodes is in the range
of mm, the geometry of the electrodes is variable. The inner diameter of the modified
tubes is 2 mm whereas the complete wall thickness is about 1 mm. The gas flow is typi-
cally in the range of 12 slm argon with up to 400 standard cubic centimeters per
minute (sccm) nitrogen admixture and up to 50 sccm oxygen admixture. To ignite the
discharge an alternating voltage of some kHz with amplitude up to 11 kV is applied.
Furthermore, a microwave driven discharge was utilized to decontaminate the
inside and the outside of medical device test specimens. This device works at
2.45 GHz and the consumed power is up to 1.5 kW. Accordingly, the gas tempera-
ture is in the range of some thousand Kelvin with gas fluxes up to 20 slm of com-
pressed (dry) air. The distance between the microwave torch and the contaminated
test specimens is about 25 cm connected via a metal tube which cooled the plasma
activated gas (see Fig. 1.3). Hence, the gas temperature of the used exhaust gas is
about 150C. The metal tube is connected to a simple process chamber where the
contaminated test specimens, 1 m long PTFE tubes with inner diameter of 2 mm and
an outer diameter of 3 mm, are mounted. During the exhaust gas propagation
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 7
through the process chamber the gas temperature further reduces. So, bacterial
inactivation induced by hot gas treatment can be obviated. The process chamber
was kept closed for 30 min, afterwards the exhaust gas was pumped down.
APPJs are very easy to handle discharge setups. Since these plasma jets are really tiny
sources of some cm length it is possible to arrange them in a very tight manner. So there
is a potential treatment of the outer surface of many different medical devices with
varying diameters from some mm up to several cm. Depending on the diameter of the
medical device the amount of APPJs has to be adjusted to guarantee a homogeneous
treatment of the whole surface. In Fig. 1.4ad some feasible arrangements of these
plasma jets are displayed. E.g. it is possible to build a ring-like alignment with different
diameters to treat catheters as shown in Fig. 1.4d.
Another method to homogeneously treat the outer surface of catheters is the use of
special plasma guiding discharge heads like the T-type head shown in Fig. 1.5. The
advantage of this setup is the use of only one source. Nevertheless, a homogeneous
treatment of the outer surface of small diameter medical devices is possible. To test the
inactivation rates of the plasma jets with T-type discharge head, catheters were divided
into six sections (each 6 cm long). Each section was contaminated with suspension of
Staphylococcus aureus, whereas the last section was kept untreated as a reference sam-
ple. The results for a gas flow of 20 slm with and without 0.25% admixture of air are
shown in Fig. 1.6. After treatment with both gas mixtures some sections of the catheter
are free of viable micro organisms (indicated in blue as minimum value). However, the
8 K.-D. Weltmann et al.
Fig. 1.4 Different plasma jet arrays for homogeneous treatment of the outer surface of medical
devices [8]
Fig. 1.5 Application of T-type plasma jet head towards catheters as representation for medical
devices [8]
Fig. 1.6 Inactivation rates of vegetative S. aureus in logarithmic scale for different gas mixtures
and amount of treatment repetition
Fig. 1.7 Direct treatment of material combinations (PTFE and metal tube) and large cavities with
a plasma jet [11]
On the other hand these discharges typically generate small plasma plumes. To treat
large areas special arrangements or discharge heads have to be invented. Moreover,
APPJs need high gas flows in the range of some slm. Especially, the high gas flow and
therefore the high costs are the limitation for some industrial applications.
Dielectric barrier discharges are to a maximum size adaptable to even complicated geo-
metrical structures. In Fig. 1.8a DBD in argon at atmospheric pressure is generated
inside a thin tube with an outer diameter of 2 mm using a high voltage driven inner
electrode. The material of the tube alternates between metal and PTFE in accor-
dance to the setup displayed in Fig. 1.1, whereas the length of the metal and the
PTFE tube section is 5 cm, respectively. This demonstrates that plasma treatment
of even difficult material junctions in combination with complicated geometry is
technically possible.
10 K.-D. Weltmann et al.
Fig. 1.8 Ignition of a DBD inside a tube with an outer diameter of 2 mm and different materials
Fig. 1.9 DBD in pure argon inside a commercially produced endoscopy biopsy channel [12]
In Fig. 1.9 the ignition of a DBD in pure argon inside a commercially produced
endoscopy biopsy channel is shown. Therefore, a silica glass capillary tube with an
implemented high voltage connected metal wire electrode is inserted into the biopsy
channel. The metal wire fortification at the outside of the tube acts as grounded
electrode. After flushing the biopsy channel with argon and applying a high voltage
signal atmospheric pressure plasma is generated inside the tube. The advantage of
this configuration is that the still existing fortification of the endoscope can easily be
used as grounded electrode. Hence, only an effortless modification on the endo-
scope is necessary to ignite a DBD. Beside this advantage the mechanical insertion
of the high voltage driven electrode into the biopsy channel can potentially lead to
damages of the inner tube wall or to small scratches, which enhances the attachment
of bacteria and other contaminants.
To avoid this disadvantage another plasma generation concept must be applied.
One technical realization is the bifilar helix electrode configuration as it is displayed
in Fig. 1.10. Here, a PTFE tube with a wall thickness of 0.5 mm is helically sur-
rounded by a pair of isolated copper electrodes.
For plasma ignition the tube is permanently flushed by a mixture of 1.5 slm
argon and 20 sccm nitrogen and a sinusoidal voltage of 20 kVpp with a frequency
of 7 kHz is applied. For endoscope implementation such high voltage amplitude
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 11
Fig. 1.10 Technical realization of a bifilar helix discharge (a) PTFE tube with a helically arranged
pair of isolated copper electrodes (b) plasma generation at atmospheric pressure in the same tube,
gas mixture: 1.5 slm argon +20 sccm nitrogen, supply voltage and frequency: 20 kVpp and 7 kHz
median (N=3)
4 minimum surviving CFU
log10 (CFU/sample)
2
detection limit
0
pure argon 20 sccm N2 10 sccm N2 0.5 slm air
+ 2 sccm O2
gas admixture
Fig. 1.11 Microbicidal efficacy of a DBD, generated by using a bifilar helix electrode configura-
tion. The surviving CFU per sample for the inactivation of B. atrophaeus spores and 0.3% BSA is
displayed for a gas flow rate of 1.5 slm pure argon and three different admixtures of nitrogen and
oxygen
might be critical for the sensitive electronics implemented into most endoscopes
(e.g. video-chip at the distal end). However, by changing the electrode shape and
reducing the isolation thickness comparable discharges with peak-to-peak voltages
below 4 kV can be generated.
The advantage of the bifilar electrode configuration is the simple plasma ignition
and the maintained tube flexibility, which is important for implementation as biopsy
channels in real endoscopes. Furthermore, different gas mixtures are selectable in
this setup.
This has an influence on the inactivation of bacteria as Fig. 1.11 indicates. The
inner tube walls (tube length: 30 cm) are contaminated with Bacillus atrophaeus
12 K.-D. Weltmann et al.
Compared to plasma jets and DBDs microwave driven discharges are mostly free
of electrodes. The temperature inside the discharge is typically in the range of
some thousand Kelvin and therefore not suitable to decontaminate heat sensitive
medical devices in direct contact (see Fig. 1.12). Here, the afterglow plasma the
exhaust gas or the plasma activated gas was used to inactivate B. atrophaeus
spores.
The test specimens were 1 m PTFE tubes with an inner diameter of 2 mm and an
outer diameter of 3 mm. The inner walls of the tubes were contaminated by rinsing
a B. atrophaeus spores solution with about 108 CFU/ml for 15 min through the
tube [13]. The outside of the tubes was spot contaminated with 100 ml suspension
of about 107 CFU/ml. The contaminated tubes were fixed in a 1.1 m long process
chamber with inner diameter of 5 cm. The process chamber was connected to the
discharge via a metal tube which cooled the plasma activated gas down to 150C.
Inside the process chamber the temperature of the gas is further reduced.
Measurements with a mass spectrometer (model: GSD 301 O1, Pfeiffer Vacuum,
Germany) on the outlet of the metal tube showed different reactive nitrogen and
oxygen species as active components.
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 13
Fig. 1.13 Inactivation results of B. atrophaeus spores after 30 min exposure time of plasma acti-
vated gas generated by a microwave driven discharge
The inactivation results for the in- and outside of the contaminated tubes after
plasma activated gas treatment for 30 min exposure time are shown in Fig. 1.13.
Obviously, the plasma activated gas shows high inactivation rates of 4.5 log10 CFU
for the inner tube walls and of 4 log10 for the outer tube walls. These results were
achieved without any alternating pressure techniques. The plasma activated gas
reaches the inside of the tube per diffusion.
In conclusion, microwave driven discharges show huge capability for decon-
tamination of medical devices. Because of the electrodeless ignition and the use of
plasma activated gas they are capable to treat even complex devices. The plasma
activated gas can penetrate into small cavities and is therefore especially interest-
ing for medical devices with fine lumina e.g. endoscopes or catheters. The device
presented in this paper uses air as process gas which in fact is very cost effective.
14 K.-D. Weltmann et al.
1.4 Conclusion
Medical devices have a complex shape and are composed of heat sensitive compo-
nents. Thus, only a small choice of conventional sterilization processes like gas
sterilization can be applied to reprocess them. Since these methods work with dan-
gerous and health hazardous chemicals, special safety conditions and/or desorption
times have to be considered. Alternatively, atmospheric pressure plasma sources
can be used to decontaminate medical devices. In this paper we presented three dif-
ferent plasma sources, namely the plasma jet, the dielectric barrier discharge and the
microwave driven discharge. Each source has its advantages and disadvantages
depending on the field of application. Plasma jets are most capable for fine lumina
and cavities and show high inactivation rates for short exposure times. Since plasma
jets are very small sources, they can be arranged in lots of different configuration to
treat large areas in short times. DBDs are advantageous concerning their spatial
dimensions. They can be designed for nearly every configuration, as shown in this
paper with the bifilar helix electrode arrangement, and work with very small process
gas fluxes. DBDs can be used in direct or indirect mode and in combination with
their high inactivation rates for short exposure times DBDs are very versatile for
decontamination of complex medical devices. The microwave driven discharges
commonly work with high electrical power and therefore generate high gas tem-
peratures. This makes most of them improper for direct treatment of medical devices.
However, the produced plasma activated gas can be used in indirect mode. The
activated gas penetrates even small lumina within short times and shows high inac-
tivation rates. This makes microwave driven discharges especially interesting for
complete sterilization of complex medical devices.
In a modern view of microbiological safety of medical products, there are no
longer processes needed for final sterilization or decontamination of the finished
product but techniques which can be introduced into methods of production as well
as reprocessing to produce a device which is safe for the designated use. The main
advantage of atmospheric-pressure plasma-based decontamination techniques is the
possibility to adapt it to special product as well as process requirements. This is the
main chance to use plasma into the medical and pharmaceutical practice [9].
Acknowledgments The work was founded by the German Federal Ministry of Education and
Research (BMBF), project name: PLASMOSE Plasmagesttzte Oberflchenmodifizierung
mittels modularer selektiver Plasmaquelle, contract number 13N8666 and: ENDOPLAS
Inaktivierende Mikroplasmen zur Sterilisierung im Lumen von medizinischen Instrumenten, contract
number 13N9320. The authors thankfully acknowledge U. Schnabel and L. Kantz for microbiological
assistance, Dr. M. Stieber and Dr. R. Brandenburg for fruitful discussions.
1 Atmospheric Pressure Plasmas for Decontamination of Complex Medical Devices 15
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PLASMOSE antimicrobial effects of modular atmospheric plasma sources. GMS Kranken-
haushyg Interdiszip 3(1):212
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tions to meet this claim? Plasma Processes Polym 5:534539
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(2007) Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure
rf-driven plasma jet. Contrib Plasma Phys 47:7279
11. Weltmann K-D, Brandenburg R, von Woedtke T, Ehlbeck J, Foest R, Stieber M, Kindel E
(2008) Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pres-
sure plasma jets (APPJs). J Phys D Appl Phys 41:194008
12. Schnabel U, Maucher T, Khnlein J, Volkwein W, Niquet R, Trick I, Stieber M, Mller M,
Werner H-P, Ehlbeck J, Oehr C, Weltmann K-D (2011) Multicentre trials for decontamination
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(2011) Assembly of standardized test specimen for microbial quantification of plasma steril-
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Chapter 2
Characterization of Damage to Bacteria
and Bio-macromolecules Caused by (V)UV
Radiation and Particles Generated by
a Microscale Atmospheric Pressure Plasma Jet
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 17
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_2, Springer Science+Business Media B.V. 2012
18 J.-W. Lackmann et al.
2.1 Introduction
The concept of separation of plasma radiation and reactive particles is based on the
fact that convection dominates the transport of heavy particles. Typical diffusion
times through the channel are orders of magnitude longer than the average residence
time of particles within the jet. For that reason the gas flow emanating from the jet
axis can be diverted by a second lateral He gas flow as long as laminar flow condi-
tions are preserved. A structure of two gas channels crossing each other in a
45-degree angle has been constructed to maintain well-controlled conditions and to
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 19
Fig. 2.1 Geometry of the X-Jet (a) Photograph of an atmospheric pressure microplasma jet in He/
O2 gas mixture with an additional He flow crossing the plasma effluent. The additional helium flow
steers the flow of radical species into a side channel. (V)UV radiation propagates along the line of
sight with the plasma through the helium atmosphere and exits the jet through the direct channel.
(b) Fluid dynamics simulation of the flow in the X-Jet. Arrows correspond to gas velocity and the
grey shading represents the flow of reactive species (e.g. O atoms). This simulation illustrates the
separation of (V)UV and heavy reactive particles
prevent admixture of surrounding gas into the gas flow (Fig. 2.1a). The modified
microplasma jet is called X-Jet. A simulation of heavy particle density confirms that
the particles are diverted into the side channel as shown in Fig. 2.1b. It is a 2D
simulation of the flow of helium (no slip boundary condition at the wall, flow 1.4 slm
in both channels) and the concentration of some representative reactive particles
from the plasma. The reactive particles react in the gas phase with a time constant
of 0.12 ms (reaction time of O with 0.2% O2 in He), it is assumed that they have a
surface reaction probability equal to 1 (approximated by setting the concentration at
the wall to 0), and that they have a binary diffusion coefficient of D = 1 cm2 s1.
The velocity field (white arrows) and the color-coded concentration of reactive
particles (from blue to red) are shown. An absolutely calibrated emission spectrum
of the jet has been measured in the past down to the wavelength of 115 nm, the
cutoff limit of the MgF2 window [4]. Two atomic oxygen emission lines at 115
(1D1D0) and 130 nm (3P3S0) dominate the spectrum. Additionally, weak emission
due to parts of the Schumann-Runge bands of O2 and a weak H line at 120 nm
(2P2S0) have been observed. We also expect that the emission spectrum contains
the He*2 excimer continuum in the 58100 nm range and a strong atomic oxygen
line at 98 nm since these emission features have been observed by other authors in
atmospheric pressure plasmas with helium [5].
It was checked that less than 0.7% of radiation between 115 and 875 nm is detect-
able on the axis of the side channel. We expect the same for the VUV radiation below
115 nm. Some reactive or excited particles can diffuse from the plasma effluent into
the direct channel when the same gas flows are used in the direct and side channels.
20 J.-W. Lackmann et al.
However, these particles will not reach the center of the direct channel after the
crossing point due to the slow diffusion and will therefore not reach the treated
surface. The flow pattern close to the surface makes sure that only the particles close
to the axis of a gas channel reaches the surface as we have shown previously [6].
Hence radiation is effectively separated from radical species. Both plasma radiation
and heavy reactive particles (mainly O3 molecules or O radicals) can be now used
separately for the treatment. The effects of (V)UV radiation treatment, reactive oxy-
gen species treatment and combined treatment of both plasma components on E. coli
and B. subtilis cells can be studied with this device.
The performance of the X-Jet was tested by treating a B. subtilis layer. The substrates,
LB agar plates with B. subtilis monolayers, were prepared as follows. B. subtilis 168
was grown for 18 h overnight at 37C in liquid LB medium [7]. The cultures were
diluted to an optical density of 0.1 at 500 nm and were applied by a 1 s spray pulse
onto LB agar plates. Before plasma treatment, the plates were incubated for 2 h at
37C. After plasma treatment, the sample plates were incubated overnight for 18 h
at 37C to allow survivors to multiply. Plasma treatment of agar plates prior to the
application of cells had no effect on bacterial growth and the pH of the medium did
not change during plasma treatment.
The agar plates were treated in air as ambient atmosphere. The jet-to-substrate
distance was adjusted to 4 mm with the direct channel parallel to the surface nor-
mal (Fig. 2.2). First, the effect of the combined treatment was tested without an
additional He flow. A typical dose-effect relationship was observed. Extending
treatment time resulted in increasing inhibition zones. When the additional He flux
through the angular channel was activated, two separate inhibition zones appeared.
A small inhibition zone approximately 2 mm in diameter formed directly under the
direct channel due to exposure to (V)UV radiation (position B in Fig. 2.2), whereas
heavy reactive species were diverted from the jet axis and caused a larger second
inhibition zone (position C). No inhibition zone(s) were observed after a 1-min
plasma treatment with either the (V)UV or particle channel indicating higher
efficiency of the combined treatment. Similar effects were observed when treating
E. coli [6].
The inhibition zone under the direct channel was larger than the 2 mm diameter
of the irradiated area. Most probably, the ambient air diffuses into the effluent under
the direct channel. The (V)UV radiation dissociates O2 molecules from air and inac-
tivation occurs on larger than expected area.
The X-Jet (1.4 slm He with 0.6% O2 and URMS = 230 V) with additional He flow
(1.4 slm) through the side channel was used for all following studies in this article.
The jet was positioned as indicated in Fig. 2.2b for the treatment with plasma radiation
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 21
Fig. 2.2 Inhibition zones after treatment of B. subtilis without and with additional He flow. The
position of the sample relative to the X-Jet is indicated on the left side with the areas treated by the
direct and the side channels indicated with letters. Without an additional He flow (a) particles and
UV migrate through the direct channel onto the plate with B. subtilis at position A. When the He
flow is turned on (b) only (V)UV propagates through the direct channel at position B, whereas
particles are steered into the side channel and treat the plate at position C
only (we will call this (V)UV channel) and it was turned by 45 to make the side
channel perpendicular to the surface for the treatment with heavy reactive particles
only (we will call this particle channel). The jet-substrate distance was always 4 mm
and the gas flow from the channel, which was not used, was blocked in such a way
that it could not reach the treated surface.
In addition to treatment of living bacterial cells, the impact of plasma and its compo-
nents on bio-macromolecules is investigated. Like all organisms, bacterial cells consist
of a lot of different macromolecules that are essential for life. In bacteria the genetic
information is stored in the nucleoid in form of double stranded DNA. In case of E. coli
and B. subtilis, the genetic information is located on a single circular chromosome.
Genes are transcribed into single stranded RNAs, which are used as templates for pro-
tein synthesis in a process called translation. Proteins fulfill structural and enzymatic
functions and are therefore the main drivers of cellular processes. Disruption of this
22 J.-W. Lackmann et al.
Plasma is capable of inducing single and double strand breaks in plasmid DNA in
liquid solution [8]. We demonstrate here that plasma also leads to the formation of
multimers in dried plasmid DNA and the formation of thymine dimers in DNA.
Aliquots of commercially available plasmid DNA (5 mg pUC18 plasmid DNA
from Fermentas, St. Leon-Rot, Germany) dried onto glass slides under vacuum
(spot size < 2 mm) were treated for 15 min either with the (V)UV channel or the
reactive particle channel of the plasma effluent. The treatment was performed under
a He atmosphere to exclude admixture of oxygen or nitrogen molecules from ambi-
ent air, which could result in absorption of the (V)UV radiation, generation of new
radicals in dissociation of ambient molecules, and destruction of plasma generated
radicals in reactions with ambient molecules. After the treatment, nucleotides were
removed from the glass slides by washing with DNase-free water and analyzed via
agarose gel electrophoresis [7] (Fig. 2.3). Aliquots dried on glass but not treated
with plasma served as control. pUC18 has a size of around 2.7 kilo base pairs (kb).
After gel electrophoresis, two distinct bands could be observed in the untreated
control, which is typical for plasmid DNA.
Circular DNA can either be in a supercoiled (lower band, around 1.8 kb) or
relaxed (upper band, around 3.4 kb) state. Plasmid DNA is isolated from bacterial
cells in the supercoiled state when there is no damage to its backbone structure at
any position. The relaxed state indicates a break in one of the two DNA strands. In
this state the plasmid is still intact. In the control sample, most of the plasmid DNA
is supercoiled as indicated by the higher band intensity of the 1.8 kb band. Only a
low amount of plasmid DNA is relaxed. These single-strand breaks most likely
occur during the drying process or when washing the DNA off the glass slides. After
treatment with the (V)UV component, the intensity of the supercoiled plasmid band
dropped visibly while a new band at a higher molecular weight of around 7 kb
appeared. According to the molecular weight standard, the new band most likely
contains plasmid dimers.
It is known that UV radiation can lead to multimer formation. UV radiation
induces DNA strand breaks and is also capable of cross-linking nucleotides from
different plasmids together [9]. Multimer formation could not be observed when
treating plasmid DNA with plasma in liquid solution [8].
Most likely, the plasmids have to be in direct contact with each other to facili-
tate multimer formation, which is only the case at very high molecule density.
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 23
Fig. 2.3 Plasmid DNA treated with the (V)UV or particle channel of the X-Jet. pUC18 plasmid
spotted on glass slides, treated with the (V)UV or particle channel of the X-Jet and analyzed via
agarose gel electrophoresis. The control was spotted on glass slides but not treated. The molecular
weight standard (10 kb DNA ladder, Fermentas, St. Leon-Rot, Germany) is commercially avail-
able. kb: kilo base pairs. Exposure times, brightness, and contrast were modified individually for
the different test conditions to optimize band visibility
After treatment with the particle channel, no dimer formation was observed.
However, a third band was detected ranging in size between the band of the super-
coiled and relaxed plasmid DNA. This band likely corresponds to linearized plas-
mid DNA indicating the introduction of double strand breaks into the DNA. It was
previously shown by OConnell et al. [8] that double strand breaks in plasmid DNA
correlate with the applied oxygen admixture in He plasma and, therefore, the amount
of emitted oxygen radicals. Our results corroborate this observation since we dem-
onstrate that plasma-generated reactive particles (including atomic O), without
additional UV radiation, are capable of inducing double strand breaks. Plasma treat-
ment is capable of modifying DNA by inducing DNA strand breaks as well as cross-
linking DNA. This highly artificial test system with high amounts of dried plasmid
DNA modified by plasma treatment suggests an industrial application for atmo-
spheric plasmas in inactivation of DNA contaminations on surfaces. Future experi-
ments will show to what extent DNA cross-linking and introduction of strand breaks
correlate with bacterial inactivation. They both could play a key role as they each
impair DNA replication and disrupt transcription.
To further investigate the impact of plasma on DNA, an 18-thymine oligomer
was treated with the different effluent channels and investigated by Fourier trans-
form infrared (FTIR) spectroscopy (Fig. 2.4a). Purified oligomers of 18 thymines
(dT18) were purchased (Thermo Scientific, Bonn, Germany) and dissolved in
A. dest. at a concentration of 0.5 mM. 20 ml aliquots were spotted on glass slides,
dried, and treated with the (V)UV or particle channel of the X-Jet in a He atmo-
sphere. Samples were measured by FTIR spectroscopy before and after treatment
and difference spectra were calculated.
24 J.-W. Lackmann et al.
Fig. 2.4 FTIR difference spectra of dT18 (a) and thymine dimer formation (b) difference absorption
spectra were calculated by subtracting the pre-plasma treatment absorption spectra from the post-
plasma treatment absorption spectra. Wave numbers for C=C and CC bonds were taken from [10]
Samples treated with the (V)UV channel for 5 min show an intensity loss around
a wave number of 1,600 cm1. Furthermore, an intensity increase is observed in the
CC fingerprint region of thymine at wave numbers less than 800 cm1. On the other
hand, samples treated for 5 min with the particle channel showed no difference in
intensities at wave numbers between 2,000 and 400 cm1. An intensity decrease at a
wave number around 1,600 cm1 indicates loss of C=C double bonds, while at the
same time, an intensity increase in the finger print region between 600 and 400 cm1
indicates the formation of CC single bonds. Both the decrease in C=C double
bonds and an increase of CC single bonds in (V)UV-treated dT18 samples, are in
accordance with thymine dimer formation (Fig. 2.4b), a well-known UV-induced
type of DNA damage. The C=C double bonds of two adjacent thymines are broken
and a cyclic butane ring consisting of four CC single bonds is formed, linking the
neighboring nucleotides covalently [11].
Thymine dimer formation is a type of DNA damage, which commonly occurs in
nature. It interrupts DNA replication and is, for instance, one of the reasons for sun-
burn and skin cancer [12]. Several bacterial cells feature highly specific DNA repair
systems, the photolyases. These enzymes use the visible part of the spectrum of sunlight
to reconstitute the double bonds [13]. In E. coli, the photolyase system is encoded by
phrB, a gene only expressed in the presence of thymine dimers [14]. This specific gene
regulation allows us to use a reporter gene fusion consisting of the regulatory region
of the phrB gene and the reporter gene lacZ encoding an enzyme, b-galactosidase, whose
enzymatic activity serves as a surrogate read-out for the gene expression level. Enzyme
activity is typically measured by conversion of a colorless substrate, e.g. 5-bromo-
4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), into a dye. The phrB-lacZ
fusion provides a means to investigate the relative level of thymine dimer formation in
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 25
Fig. 2.5 phrB-lacZ fusion treated with the UV or the particle channel overnight. E. coli mutants
were plated on LB agar plates containing X-gal as substrate and were treated with plasma compo-
nents overnight. Controls were treated simultaneously with gas only. Blue color (area marked by
dotted line) indicates expression of phrB. Only the plate treated overnight with the (V)UV-channel
shows blue color directly under the jet nozzle (details of agar plates were magnified). Color figure
available in online version
the living bacterial cell, in vivo, by comparing b-galactosidase activity under control
growth conditions and after plasma treatment.
An E. coli mutant carrying the reporter gene fusion was treated with the different
X-Jet channels (Fig. 2.5) and gene expression, and, therefore, indirectly thymine
dimer levels, were monitored by the amount of X-gal converted into a blue dye [15].
To this end E. coli DH5a cells carrying the phrB reporter gene fusion in addition
to the phrB gene were incubated overnight at 37C in LB medium with tetracycline
to select for the reporter plasmid. In order to achieve a monolayer of bacterial cells
on LB agar plates containing X-gal and tetracycline, the overnight culture was
diluted to an optical density of 0.1 at 580 nm and applied to the plates in a 1-s spray.
These LB agar plates were incubated for 2 h at 37C before treatment with short
plasma pulses (1 s plasma every 180 s) overnight at room temperature. This mild
long-time treatment ensures continuous but non-lethal exposure to plasma-caused
stress, requiring the cells to cope with the inflicted damages by inducing repair
mechanisms, while still allowing cell proliferation. Either the (V)UV or particle
channel was used for treatment and an additional control was treated with He/O2 gas
mixture only. LB agar plates were photographed (Fig. 2.5). Formation of the blue
color indicates b-galactosidase activity and, therefore, presence of thymine dimers.
The blue dye was only observed in case of treatment with the (V)UV channel, but
not after exposure to the particle channel or under control conditions. These results
indicate the formation of thymine dimers in vivo specifically by (V)UV radiation.
We can conclude from our experiments that plasma can damage DNA both
in vitro and in vivo. Whereas in vitro tests using isolated bio-macromolecules offer
26 J.-W. Lackmann et al.
Fig. 2.6 B. subtilis total RNA before and after treatment. The control sample (C) was treated with
He/O2 gas only. The other samples were treated for 2 min with the (V)UV channel or the particle
channel of the X-Jet
Little is known about plasma-induced damage to RNA molecules although this may
be another mechanism relevant for bacterial inactivation. RNA is transcribed from
a DNA template and can itself serve as a template for protein synthesis. Stable RNA
molecules play a key role as part of ribosomes, the protein synthesis factories of the
cell. To analyze the effect plasma has on RNA, B. subtilis total RNA, containing
approximately 90% stable RNA molecules, was extracted from bacterial overnight
cultures [16]. 10 mg aliquots were spotted on RNase-free glass slides and treated
with the X-Jet channels for 2 min. After treatment, RNA was washed off the glass
slides with RNase-free water and analyzed via RNA-optimized agarose gel-electro-
phoresis [16] (Fig. 2.6). The control was also dried on glass slides but only treated
with He/O2 gas instead of plasma for 2 min. The two distinct bands in the control
lane are the stable 23S and 16S rRNAs that make up most of the total RNA in a cell.
Intensities of these bands decreased during plasma exposure.
In contrast to the DNA treatment experiment, no RNA multimer formation was
observed after treatment with the UV or the particle channel. Under both treatment
conditions band intensity decreased, with the effect being much more pronounced
after treatment with the particle channel. Possible explanations could be RNA
2 Characterization of Damage to Bacteria and Bio-macromolecules Caused 27
When RNA is translated into an amino acid chain, this chain forms secondary and
tertiary structures to finally become a functional protein. Proteins are highly diverse and
fulfill many essential cellular functions. Destroying the functionality of a single essential
protein species is sufficient to compromise cell viability. Therefore, protein damage is
discussed as a major inactivation mechanism [17]. One well-characterized impact of
plasma on protein is etching, the removal of matter by plasma treatment [18].
BSA aliquots were applied to Si-wafers and dried under vacuum to create a plain
protein layer with a thickness of few micrometers. These layers were exposed to the
(V)UV channel or the reactive particle channel of the X-Jet (same conditions as
above) for 10 min. The treatment was performed in a controlled He atmosphere with
a jet-to-substrate distance of 4 mm. The etching profiles were determined by profi-
lometry. In this measurement, a sharp tip scans the surface topography along a
straight line, which, in this case, was a line over the symmetry axis of the etched
structure. Etching of a BSA layer, a biological model sample, was compared to the
etching of a non-biological model plasma polymer layer, a so-called hydrogenated
amorphous carbon (a-C:H) film. A 300 nm a-C:H film was produced in low pressure
CH4 plasma. The a-C:H film had an atomic hydrogen content of 45% and a density
of around 1 g cm3 [6]. The etching profile of the a-C:H film has a bell shape with a
full width at half maximum around 2 mm and with a maximum etching rate of about
30 nm/min (Fig. 2.7a). This profile corresponds perfectly to a flux of atomic oxygen
to the surface as predicted by a 2D fluid simulation of particle transport and reaction
kinetics in the effluent [6].
Similar etching profiles with the same etching rates were observed when treating
the BSA protein layer and the a-C:H film as shown in Fig. 2.7a. The profiles indi-
cate that heavy reactive particles in the plasma effluent (likely oxygen atoms) can
etch protein layers effectively and the etching mechanism does not depend strongly
on the layer structure of the sample. The measured BSA profile has much higher
roughness than the a-C:H film. This likely has two reasons, namely the inhomoge-
neity occurring in the drying process and the formation of cracks in the part of the
layer exposed to the reactive particles, which were detected by optical microscopy
(Fig. 2.7b). These cracks could be caused by tensile stress in the layer, which appears
during the etching as a result of the shrinking of the layer structure.
Both the a-C:H film and the BSA layer were also exposed to the (V)UV compo-
nent of the plasma effluent. No changes in the a-C:H film thickness or structure
were observed on the time scale of 20 min. In case of BSA, sample roughness was
increased after 20 min of (V)UV treatment suggesting minor damage of the upper
layer of the protein coat (data not shown).
28 J.-W. Lackmann et al.
Fig. 2.7 BSA and a-C:H etching with the particle channel of the X-Jet. (a) A BSA layer or an
a-C:H film were treated with the particle channel of the X-Jet under He atmosphere for 10 and
4 min, respectively. Etching rates per minute were calculated. Measured curves (4,000 points/mm)
were smoothed using a Savitzky-Golay algorithm set to 700 points. (b) An optical microscope
image shows the BSA layer after treatment with the particle channel
2.5 Conclusions
With the new X-Jet design of an atmospheric pressure plasma jet operated in He/O2
gas mixture, plasma radiation and heavy reactive particles in the effluent can be
separated effectively. The X-Jet allows to study separately the effects of (V)UV
plasma radiation and reactive particles on living cells and biological matter.
We were able to demonstrate that both plasma components inactivate vegetative
bacterial cells. DNA treatment with the (V)UV channel resulted in thymine dimer
formation both in vitro and in the living cell. Treatment with the reactive particle
channel led to introduction of DNA single and double strand breaks in vitro. We
further observed damage to stable RNA molecules in vitro after treatment with both
the (V)UV and the particle channel. Exposure of protein to the reactive particles
showed an etching rate for biological macromolecules comparable to that of an
a-C:H film. In vivo studies to investigate plasma effects on RNA and proteins inside
of vegetative bacterial cells are underway.
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6. Schneider S, Lackmann J-W, Narberhaus F, Bandow JE, Benedikt J (2011) Separation of
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Cold Spring Harbor Laboratory Press, New York
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(2011) Cold atmospheric pressure plasma jet interactions with plasmid DNA. Appl Phys Lett
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plasmid pUC19 DNA restriction fragments. Photochem Photobiol 65:945948
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Thieme, Stuttgart
11. Setlow RB (1966) Cyclobutane-type pyrimidine dimers in polynucleotides. Science 153:379386
12. Matsumura Y, Ananthaswamy HN (2004) Toxic effects of ultraviolet radiation on the skin.
Toxicol Appl Pharmacol 195:298308
13. Hearst JE (1995) The structure of photolyase: using photon energy for DNA repair. Science
268:18661872
14. Goosen N, Moolenaar GF (2008) Repair of UV damage in bacteria. DNA Repair 7:353379
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Chapter 3
Bio-Decontamination of Water and Surfaces
by DC Discharges in Atmospheric Air
3.1 Introduction
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 31
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_3, Springer Science+Business Media B.V. 2012
32 Z. Machala et al.
various plasma jets, usually in rare gases (He or Ar) with/without admixtures of O2
(or H2O). The plasma jets are typically blown into the ambient air where the rare gas
plasma entrains air components. Air plasmas at atmospheric pressure have addi-
tional advantages of no need of special gases and an easy application in ambient
environment. Various atmospheric air plasma discharges have been tested for bio-
decontamination: DC [14], AC [5], dielectric barrier (DBD) [613], RF [14], and
pulsed discharges [8, 15, 16]. The plasmas can be also generated directly in water
or on water-air boundary [1, 9, 10, 17, 18], which is of great interest for water
decontamination. Atmospheric plasmas have been tested on a large variety of
prokaryotic microorganisms, such as bacteria, spores, viruses, and some eukaryotic
yeasts, fungi and microalgae, resulting in partial disinfection (12 log reduction of
microbial population) up to complete sterilization. Interaction of the plasmas with
the tissues of higher organisms including humans and plasma applications for skin
disinfection, wound healing, blood coagulation, dentistry, surgery, inducing apoptosis
pathways for cancer treatment, etc. are targeted by a revolutionary novel discipline
plasma medicine.
In bio-decontamination by plasma, it is crucial to understand the role of various
mechanisms involved. The significant mechanisms depend on the plasma composi-
tion (gas), temperature, treated microorganisms and the environment (air, water,
surfaces, etc.). In atmospheric pressure plasmas, the major role is typically attrib-
uted to radicals and reactive oxygen species (ROS, e.g. OH, O, O3) [1, 2, 6, 1012,
1416, 1820] and to charged particles, especially O2 [7, 21] affecting the cell
membranes. UV radiation plays a role only if photons in UV C germicide region
(220280 nm) or in vacuum UV are produced [11, 18]. In cold air discharges
(corona, DBDs, pulsed discharges), UV C or VUV are usually not generated, so
radicals and ROS are identified as the dominant bio-inactivation agents [1, 6, 9, 10,
15, 20, 22, 23].
Our former bio-decontamination investigations with DC discharges in atmo-
spheric air with water were in agreement with this statement and demonstrated the
dominant role of radicals and ROS [24, 25]. In this paper, the biocidal effects of two
atmospheric air plasma sources treating water and surfaces are investigated positive
DC streamer corona (SC) and transient spark (TS). Despite DC applied voltage,
these discharges have a pulsed character with nanosecond repetitive pulses. The
pulsed plasmas are especially convenient for penetration into topographically non-
uniform surfaces and cavities, which is applicable e.g. in teeth root canal disinfection
[6, 8, 16, 26].
The effects of SC and TS on selected bacteria in water solutions and on various
surfaces were tested. Bio-decontamination of water is important for waste water
cleaning or drinking water disinfection and is involved in all bio-medical applica-
tions and food technology, since cells and most foods contain water. Plasma decon-
tamination of various surfaces is important in medicine, e.g. for the sterilization of
endoscopes, implants and other heat sensitive materials, and in dentistry for cavities
or dental plaque treatment [27, 28] or root canal disinfection [6, 8, 16, 26].
We focus on the identification of the dominant plasma agents in bio-inactivation
by coupling the electrical characteristics, emission spectra, and biocidal effects of
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 33
the studied discharges in various regimes with the measurements of the oxidative
stress induced in microbial cells. Comparing direct with indirect plasma effects
enables further separation of various biocidal plasma agents (electric field, charged
particles, neutral active species, UV radiation).
The discharge set-up shown in Fig. 3.1 enabled the contaminated water to flow
directly through the high voltage hollow needle electrode, and so through the
corona active region in its proximity. The effect of electrostatic spraying occurred
when the high voltage was applied on the needle electrode [29]. Figure 3.2 shows
the photographs of the electro-spray of water with SC and TS discharges. These
experiments were repeated 510 times for each discharge type at various operation
parameters.
34 Z. Machala et al.
Fig. 3.1 Experimental setup for DC discharges, with a high voltage hollow needle electrode
enabling water flowing through the discharge zone and a plane or mesh electrode
Fig. 3.2 Photographs of the electro-spray of water in 8 mm gap, water flow rate 0.5 mL/
min, exposure time 1/10 s: (a) electro-spray with SC, 6.5 kV, (b) transition SC-TS, 7.8 kV,
( c) TS, 9 kV
We compared direct and indirect plasma effects on contaminated solid agar sur-
faces. A needle electrode was placed about 1 cm above the agar surface in the
centre of the Petri dish and the SC or TS was applied for various treatment times
(5 s2 min). In direct treatment, the agar was grounded with a wire. Indirect plasma
effects on the contaminated agar were tested by placing the grounded mesh elec-
trode ~2 mm above the agar; this shielded the electric field and expectantly trapped
the charged particles, letting but neutral particles and partial UV light to reach the
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 35
Fig. 3.3 Schematics of electrode arrangements for (a) direct and (b, c) indirect plasma treatment
of contaminated agar plates. (b) Mesh electrode ~2 mm above agar surface trapped the charged
particles and shielded the electric field. (c) Quartz (or MgF2) window only transmitted light from
the discharge (including UV)
The plastic and teeth surfaces were treated by positive and negative corona only. TS
resulted in the material damage after a few minutes of treatment. Figure 3.4 shows
the photographs of plastic foils and extracted human teeth (provided by volunteers)
treated by positive and negative DC corona. The foils and the teeth samples were
placed on the plate grounded electrode and the high voltage was applied to the needle
5 mm above them. We compared treatment of dry and moist foils. The teeth were
always treated moist (physiological solution) to mimic their natural environment.
Fig. 3.4 Photographs of contaminated plastic (a, d) and tooth (b, c, e, f) samples with atmospheric
pressure air DC discharges: 1st row positive streamer corona, 2nd row negative corona (Trichel
pulses)
per dish. Sterile polypropylene foils and extracted human teeth were contaminated
by B. cereus spores (20 mL drop with about 104105 spores).
The microbial cultivation was carried out in a sterile environment in the follow-
ing steps: an overnight bacterial culture was first prepared in a shaker with sterile
liquid nutrient. A hot sterile nutrient medium agar was poured into sterile Petri
dishes, on which the bacteria were grown, and solidified. Cultivated bacteria in the
liquid nutrient were compared with McFarland turbidity scale to assess their initial
population per mL. They were then diluted in water to obtain desired concentra-
tions. The plasma experiments were performed with both discharges, at various
parameters and treatment times and repeated 510 times. Three to five Petri dishes
from each sample were taken for statistical evaluation. These were incubated during
1224 h in a thermostat at 37C. The grown CFUs on the treated and reference
samples were counted and evaluated.
Interaction of ROS with the bacterial cell membranes results in the peroxidation of
membrane lipids. The final product of lipoperoxidation is malondialdehyde (MDA),
quantifiable by spectrophotometry after the reaction with thiobarbituric acid (TBA)
at 90100C [30]. This method of thiobarbituric acid reactive substances (TBARS)
was applied to measure the oxidative stress induced in bacteria in distilled water
exposed to SC and TS similar to [24, 25]. We assigned the TBARS concentrations
from the absorbance of MDA at 532 by using Lambert-Beers law with the absorp-
tion coefficient 1.57 105 mol1 L cm1 [30].
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 37
a 25 b 20
18
20 16
U [kV] U [kV]
14
U [kV] / I [mA]
I [mA] I [A]
U [kV] / I [A]
12
15
10
8
10
6
4
5 2
0
0 2
100 0 100 200 300 400 40 20 0 20 40 60 80 100
t [ns] t [ns]
Fig. 3.5 Typical voltage and current waveforms of positive (a) SC and (b) TS discharges with
electro-spray of water, 10 mm gap
The water solution contaminated by bacteria (B. cereus and S. typhimurium) flew
directly through the high voltage hollow needle electrode, and so through the plasma
active zone in its proximity. In addition, the effect of electrostatic spraying substan-
tially improved the efficiency of bio-decontamination compared to our previous
set-ups for water treatment [24].
The efficiency of transient spark was higher than of streamer corona. TS decreased
the initial microbial population roughly by 3 logs and SC by 1 log. We use a new
parameter E-value (Joule per treated water volume and one log reduction of microbial
population) to express the combined energy requirements and efficiency of the pro-
cess. Streamer corona is far less energy-demanding than TS, as shown in Fig. 3.6.
The temperature of the treated water did not change in SC and was increased by
maximum 10 K in TS. The lethal heat effect of the discharges to bacteria can be
excluded.
The chemical effects induced in the plasma treated water were measured by pH and
conductivity probes and spectrophotometric method for H2O2. Depending on the
initial conductivity of the treated water (1, 500, 1,000 mS/cm, physiologic solution
~14 mS/cm) and the plasma parameters, we observe a pH decrease from 57 down
to 35 and an increase of conductivity (from 1 up to 1,000, from 500 up to 1,300 mS/cm).
The measured peroxides (H2O2) reached up to 500 mM. pH decrease is probably due
to the nitric acid formation. However, additional tests showed that the nitric acid
solution of the same pH does not lead to the same biocidal effects. In agreement
with [34], it is clear that acid environment in synergy with plasma agents leads to
the bacterial inactivation. In addition, we suppose an interaction of nitrites and
peroxides at lowered pH; this has to be further studied with using buffers.
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 39
Efficiency Evalue
100
90
10
85
80
1
75
TS+ SC+ TS- SC-
Fig. 3.6 Comparison of inactivation efficiency of B. cereus in saline solution with E-value for
positive and negative TS and SC. Medians with 1st and 3rd quartiles as error bars. A typical num-
ber of repeated experimental sets was 10
Figure 3.7 shows the inactivation efficiencies for both positive discharges applied to
the electro-sprayed water with the measured concentrations gains Dc(TBARs) of
TBARs for B. cereus and S. typhimurium. The same bacterial samples were irradiated
by biocidal UV C radiation (Hg lamp, 254 nm, 1 min) for comparison. Concentrations
c(TBARs) correlated with the inactivation efficiencies of the discharges.
UV C radiation induced almost no Dc(TBARS) despite its efficiency was very
high. Obviously, UV dominant biocidal mechanism is not peroxidation of cell mem-
branes. On the contrary, SC and TS plasma treatments significantly enhanced the
TBARS concentration. This indicates that oxidations of cell membranes by reactive
oxygen species ROS are important in microbial inactivation.
c(TBARS) [mol/l]
0.06 94 94
Efficiency [%]
Efficiency [%]
92 0.10 92
90 90
0.04 0.08
88 88
86 0.06 86
0.02 84 0.04 84
82 82
0.02
80 80
0.00
78 0.00 78
TS+ SC+ UV TS+ SC+ UV
Fig. 3.7 TBARS concentration gains (with respect to control) and decontamination efficiencies of
B. cereus and S. typhimurium in water treated by SC and TS with electro-spray, compared with
30 s exposure to UV C (Medians with the 1st and 3rd quartiles as error bars; n is the number of
repeated experimental sets)
Fig. 3.8 Photographs of contaminated agar surfaces with area inactivated by direct (upper row)
and indirect (lower row) exposure to TS for various exposure times. 60 s (far right) is not in the
same scale
The heat effect of TS on contaminated agar is possible in the very small area
under the discharge. TS usually resulted in a tiny hole (12 mm diameter) of dried
agar. Nevertheless, the area of decontamination (void) was always much larger than
this tiny hole in its centre. The void kept the ambient temperature after treatment, so
we can neglect the heat effect on bacteria. In the indirect treatment, the TS heat was
lead out through the mesh electrode which was not in the direct contact with the
agar surface: the heat effect can be thus excluded.
Figure 3.9 shows the inactivated area (normalized through three experimental
series) for direct and indirect exposure for short treatment times (515 s). The
inactivated area increases with the exposure time. The direct exposure is much
3 Bio-Decontamination of Water and Surfaces by DC Discharges in Atmospheric Air 41
direct
95%
75%
median
25%
2 5%
0
5 10 15
treatment time [s]
Fig. 3.9 Normalized inactivated area as a function of treatment time for direct and indirect exposures
to TS. Medians, 1st and 3rd quartiles, and 5th and 95th percentiles; statistics of 3 repeated experimental
series, 2 3 5 samples each
stronger for 5 s, which indicates that charged particles are important at the beginning
of the treatment. However, at 15 s the direct and indirect effects become more
similar. At 60 s treatment, there is very little difference between the direct and
indirect exposures with both discharges, indicating a crucial role of reactive neu-
tral species.
Exposure to the UV light only transmitted by quartz or MgF2 windows demon-
strated no visible decontamination. This correlates with the emission spectra of SC
and TS lacking UV C or VUV light.
Plastic foils (dry and moist) and human teeth surfaces (moist) contaminated with
B. cereus spores were treated by positive and negative streamer corona. Figure 3.10
shows the results in efficiency vs. energy graphs. The positive SC was more effective
and more energetic on dry plastic samples, whereas the negative pulsed corona was
more efficient on moist ones with about the same energy consumption. Negative pulse-
less corona was largely more energetic (~247 J) despite it provided the best sporicidal
efficiency (~93%). The experiments with moist teeth surfaces showed that the efficien-
cies in 3 and 5 min exposure times were almost the same (within the error bars). This
suggests that in this set-up, it does not make sense to extend the treatment time. Positive
streamer corona was the most efficient and negative Trichel pulse corona slightly less
42 Z. Machala et al.
Fig. 3.10 Decontamination efficiency vs. energy (medians with first and third quartiles as error
bars): dry or moist plastic foils 2 min treatment (left, 3 experimental sets with 46 samples each)
and moist teeth 3 and 5 min treatment (right, 3 experimental sets with 57 samples each)
efficient and less energetic. These preliminary findings are now being verified in the
treatment of teeth contaminated by Streptococcus mutans biofilms.
3.4 Conclusions
Acknowledgments Effort sponsored by Slovak grant agency VEGA 1/0668/11 and 1/0711/09,
Slovak Research and Development Agency APVV SK-CZ-0179-09. The human teeth were pro-
vided by patients of Dr. O. ipoldov. We thank P. Luke (IPP Prague) for his motivating ideas, and
I. Jedlovsk and B. Pongrc (FMFI Bratislava) for assistance.
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species in N2O2 flowing post-discharges at atmospheric pressure for sterilization. J Phys
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16. Jiang C, Chen MT, Gorur A, Schaudinn C, Jaramillo DE, Costerton JW, Sedghizadeh PP,
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Polym 6:479
17. Mizuno A, Hori Y (1988) Destruction of living cells by pulsed high-voltage application.
IEEE Trans Ind Appl 24:387
18. Luke P, lupek M, Babick V, unka P (2008) Ultraviolet radiation from the pulsed corona
discharge in water. Plasma Sources Sci Technol 17:024012
19. Brandenburg R, Ehlbeck J, Stieber M, Woedtke Tv, Zeymer J, Schluter O, Weltmann KD
(2007) Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure
rf-driven plasma jet. Contrib Plasma Phys 47:72
20. Gweon B, Kim DB, Moon SY, Choe W (2009) Escherichia coli deactivation study controlling
the atmospheric pressure plasma discharge conditions. Curr Appl Phys 9:625
21. Dobrynin D, Fridman G, Friedman G, Fridman A (2009) Physical and biological mechanisms
of direct plasma interaction with living tissue. New J Phys 11:115020
22. Lu X, Ye T, Cao Y, Sun Z, Xiong Q, Tang Z, Xiong Z, Hu J, Jiang Z, Pan Y (2008) The roles
of the various plasma agents in the inactivation of bacteria. J Appl Phys 104:053309
23. Laroussi M (2005) Low temperature plasma-based sterilization: Overview and state-of-the-art.
Plasma Processes Polym 2:391
24. Machala Z, Jedlovsk I, Chldekov L, Pongrc B, Giertl D, Janda M, ikurov L, Polic P
(2009) DC discharges in atmospheric air for bio-decontamination - spectroscopic methods for
mechanism identification. Eur Phys J D 54:195
25. Machala Z, Chldekov L, Pelach M (2010) Plasma agents in bio-decontamination by dc
discharges in atmospheric air. J Phys D Appl Phys 43:222001
26. Lu X, Cao Y, Yang P, Xiong Q, Xiong Z, Xian Y, Pan Y (2009) An RC plasma device for
sterilization of root canal of teeth. IEEE Trans Plasma Sci 37:668673
27. Sladek REJ, Stoffels E, Walraven R, Tielbeek PJA, Koolhoven RA (2004) Plasma treatment of
dental cavities: a feasibility study. IEEE Trans Plasma Sci 32:15401543
28. Yu QS, Huang C, Hsieh F, Huff HE, Duan YX (2007) Bacterial inactivation using a low-
temperature atmospheric plasma brush sustained with argon gas. J Biomed Mater Res B Appl
Biomater 80:211219
29. Borra J-P, Ehouarn P, Boulaud D (2004) Electrohydrodynamic atomisation of water stabilised
by glow discharge - operating range and droplet properties. J Aerosol Sci 35:1313
30. Bachowski GJ, Pintar TJ, Girotti AW (1991) Photosensitized lipid-peroxidation and enzyme
inactivation by membrane-bound merocyanine-540 - reaction-mechanisms in the absence and
presence of ascorbate. Photochem Photobiol 53:481
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(2007) Emission spectroscopy of atmospheric pressure plasmas for bio-medical and environ-
mental applications. J Mol Spectrosc 243:194
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transitions. IEEE Trans Plasma Sci 36:918
33. Janda M, Martiovit V, Machala Z (2011) Transient spark: a dc-driven repetitively pulsed
discharge and its control by electric circuit parameters. Plasma Sources Sci Technol
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The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids.
Plasma Processes Polym 7:250
Chapter 4
Biological Decontamination Using Pulsed
Filamentary Microplasma Jet
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 45
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_4, Springer Science+Business Media B.V. 2012
46 R. Pothiraja et al.
4.1 Introduction
Atmospheric pressure cold plasmas are very useful for the production of UV
photons, excited atoms/molecules, radicals and charged species. Although these
active species can also be produced in low pressure plasmas as well as in ther-
mal plasmas, using atmospheric pressure cold plasmas has many advantages.
Since it is operated at atmospheric pressure, there is no need for any vacuum
equipment and makes the device portable. In addition to this, despite the high
temperature of the electrons, the average gas temperature is close to room tem-
perature because of the low energetic ions; hence, suitable for the treatment of
temperature sensitive objects without affecting their bulk properties. In this
regard, plenty of research efforts are being made in order to understand and
optimize the atmospheric pressure cold plasma for the production of various
specific active species for different specific applications. One of the most impor-
tant and exciting applications of these plasmas are in the field of biomedicine
for cancer treatment [1], sterilization [2], dental bleaching [3], blood coagula-
tion [4], and wound healing [5]. As for the sterilization of microorganisms is
concerned, many sterilization techniques already exist, however, they either
involve exposure to toxic chemicals such as ethylene oxide or involve subject-
ing the specimen to high temperatures, as in the case of autoclaving. This is not
suitable for many heat sensitive and biological materials, especially for medical
and food package applications.
Low temperature plasmas operating at atmospheric pressure are particularly
well suited for treatment of these sensitive materials. In this regard, several
research groups have developed various plasma sources with a range of power
supply and electrode configurations; and studied efficiency their plasma source
towards inactivation or sterilization of various microorganisms including
Escherichia coli (E. coli) and spores of Bacillus atrophaeus (B. atrophaeus), and
studied inactivation or sterilization mechanism. For example, Laroussi et al. used
resistive-barrier discharge (RBD) to study the decontamination and its mecha-
nism [6]. Park et al. used microwave induced argon plasma for the sterilization
studies [7]. Fridman et al. used DBD based plasma source and studied the effect
of direct plasma and indirect plasma on sterilization [8]. Kong et al. used DBD
based plasma plume for the inactivation of spores [9]. Machala et al. used positive
DC discharge in water spray for the decontamination of bacteria [10]. Weltmann
et al. used plasma jet with RF power supply to study the effectiveness of plasma
against wound pathogens [11]. We have used pulsed positive filamentary plasma
source operating in coaxial DBD principle for the inactivation of E. coli (Gram-
negative bacteria) and spores of B. atrophaeus (Gram-positive bacteria). Here, we
report the plasma source configuration, plasma characterization using optical
emission spectroscopy, inactivation of B. atrophaeus and E. coli, and possible
mechanism of inactivation.
4 Biological Decontamination Using Pulsed Filamentary Microplasma Jet 47
Experimental setup used for the biological decontamination studies, and methods
used for plasma characterization are similar to the one reported previously [12].
Hence, only a brief description is given here. Our microplasma jet consists of a
tungsten electrode with the diameter of 1.6 mm. One end of this electrode is
sharpened to an angle of 30, while the other end of the electrode is connected
to a high voltage generator. The output voltage and the pulse frequency of this
generator can be controlled and varied from 0 to 20 kV and from 4 to 500 kHz,
respectively. Each high voltage pulse exhibits a sequential profile with damped
oscillations. A tube made of quartz with the inner diameter of 6 mm, is used in
order to test the decontamination of the tube. The outer diameter of the tube is
8 mm. The tungsten electrode is placed coaxially inside this quartz tube
(Fig. 4.1). A copper tube is used as a grounded electrode. Length of the grounded
electrode is about 40 mm. It is placed coaxially on the outer surface of the quartz
tube and is separated from the inner electrode. Hence, the quartz tube also acts
as dielectric in between the electrodes. The distance between the driven elec-
trode and the grounded electrode can be varied, and for the present studies, it is
kept as about 50 mm. Because of its transparency in UV-Vis region, the quartz
tube facilitates the plasma characterization through emission spectroscopy. A
void is present in the grounded electrode, through which information of the fila-
ment generated inside the quartz tube in the grounded electrode region is
obtained. Argon is used as the working gas during decontamination studies. The
flow rate of argon is kept as 2.4 slm. Nitrogen (about 1 sccm) is admixed with
argon during diagnostic studies.
Relatively and absolutely calibrated echelle (ESA 3000) and Ocean Optics
(QE65000) spectrometers are used to obtain the emission spectra [13]. The solid
angle of the optical fiber used in echelle spectrometer amounts to 0.012 sr. The
diameter of entrance hole of diaphragm is 1 mm. Spectral resolution of the echelle
spectrometer amounts to Dl = 0.015 nm at l = 200 nm and Dl = 0.060 nm at
l = 800 nm. The groove density of echelle grating, g = 75 grooves per mm. The
optical resolution of Ocean Optics spectrometer is ~0.147.7 nm. A Pearson cur-
rent monitor (model, 6585; output, 1V = 1A) is used for plasma current measure-
ment, which is mounted around the cable connecting the generator and the tungsten
electrode. The output of the current monitor is connected to an oscilloscope
(LeCroy 9314A). The actual voltage applied for plasma generation is measured by
connecting the output of the generator to the oscilloscope through a voltage divider
with the dividing factor of 2,000. The pulse frequency is fixed as 22 kHz for all the
experiments. For the plasma volume determination, a high speed sensitive camera
(PCO sensicam qe) is used.
48 R. Pothiraja et al.
Intensity (a.u)
with the experimentally
measured emission spectrum Experimental
The relative intensity of N2(CB, 00) with respect to the intensity of N2+(BX,
00) is used for the determination of electron velocity distribution function (EVDF)
by including the contribution of excitation of nitrogen molecules by argon meta-
stables. For this purpose, the relative intensity of N2(CB, 00) with respect to the
intensity of N2+(BX, 00) is simulated for various EVDFs for our experimental
conditions by numerical solution of the Boltzmann equation in local approximation
and varied electric field applying the program code EEDF developed by
Napartovich et al. [16]. Finally, by comparing the experimentally determined
I + I N+ (B- X)
relative intensities ( N2 (B- X) ) with the simulated relative intensities ( 2 )
I N2 (C - B) I N2 (C - B)
for various EVDFs, the actual EVDF is determined.
Using the normalized EVDF and the known collisional cross section sexc (cm2)
for electron impact excitation [17], we calculate the rate constants k (cm3s1) for
electron impact excitation of N2, using the Eq. 4.1:
2C
k = 4p 2 fv ( E ) E s exc ( E ) dE, (4.1)
0 me
where, me is the mass of electron (g), E is the kinetic energy of electrons (eV) and
C = 1.602 1012 erg eV1.
The electron density (ne, cm3) is determined using the Eq. 4.2 from the measured
absolute intensity of N2(CB, 00) emission ( I N2 (C - B) , photcm3s1), nitrogen den-
sity ([N2], cm3), the electron impact excitation rate constant for N2(CB, 00) emis-
sion ( kN2 (C) , cm3s1), contribution of excitation of N2(C) by collision with argon
50 R. Pothiraja et al.
metastables ( K Ar
N 2 (C) ) [12], contributions of the quenching of N2(C) by argon ( Q N 2 (C) ),
met
the plasma volume (Vp, cm3), value of fraction of time in which plasma is active (tf),
and the geometrical factor (gf), which relates the total quantity of photons produced
in the plasma source to the observed quantity of photons by the emission spectrom-
eter. To determine this geometrical factor, we consider the actual volume of plasma,
from which spectrometer receives the photons as well as the distance between the
active plasma volume and the entry point of optical fiber.
I N2 (C - B)
ne = (4.2)
[N 2 ](kN2 (C) + K NAr2met(C) )Q N2 (C) Vp t f g f
For the E. coli decontamination experiments, the E. coli DH5a strain is incubated
over night in LB media at 37C [18]. It is diluted to an optical density of 0.5 at
580 nm; 50 ml of this solution is spread onto agar plates of diameter 9 cm. Hard agar
plates are made with 30 g of Agar-Agar per liter media, while soft agar plates are
made with 15 g of Agar-Agar per liter media. After plating, the agar plates with the
bacteria are incubated for an additional 2 h at 37C. After plasma treatment, it is
incubated at 25C for 3 days. From the visible change on agar plate due to the
plasma treatment, the inhibition zone is determined. For the decontamination exper-
iment of E. coli coated tube, 5 105 cells are coated inside the tube. After plasma
treatment, the tube is washed and the survival cells are counted by usual method
[18]. For all experiments, reference samples are made and are analyzed along with
plasma treated samples.
For the decontamination of spores of B. atrophaeus, first set of experiments are
carried out with about 300 spores placed on a polyethyleneterephthalate (PET)
polymeric plate in a circular area of a few mm diameter. The second set of experi-
ments are carried out by placing about 4,700 spores placed on each PET plate in a
circular area with a few mm diameter. A standard procedure reported elsewhere is
used for the samples preparation and analysis [18].
Long plasma filaments are generated as positive leader like discharge inside a tube in
pure argon as the working gas (Fig. 4.3). Decontamination experiments are carried
out at this condition. However, during plasma diagnostic studies, nitrogen is admixed
with argon. Plasma parameters are determined at two regions in the filament; one at
4 Biological Decontamination Using Pulsed Filamentary Microplasma Jet 51
Current (A)
discharge. The pulse
0 0.4
frequency is 22 kHz. The
voltage frequency of each 3 0.2
pulse sequence is around Current
200 kHz 6 0.0
9 0.2
0.0 2.0x105 4.0x105
Time (s)
the midpoint in between the powered electrode and grounded electrode, and the
second point is at the middle region of the grounded electrode.
Gas temperature in active plasma is about 600 K. Since the discharge duration is
in the range of 100200 ns, the actual temperature will be much less than this value,
especially in the effluent of plasma. This factor gives the possibility to use this plasma
source for decontaminating the temperature sensitive materials. The power dissi-
pated is about 12 W during the discharge. The reduced electric field close to spike of
driven electrode is high (about 1,700 Td) compared to this in grounded region (about
900 Td). High reduced electric field indicates that argon ionization reaction is more
probable than the argon metastables formation reaction. The electron density in both
measured points is comparable with each other, and is in the order of 1011 cm3. This
indicates that the average ion density in active plasma is in the order of 1011 cm3,
which can play important role especially in direct plasma treatment.
The E. coli layered agar plates are placed at 11 cm coaxially away from the grounded
electrode. The exit point of effluent in the tube is placed at the centre of the agar
plate. The distance between the effluent exit point of the tube and the agar plate is
about 2 cm. This inhibition studies are carried out for E. coli on soft agar plates as
well as on hard agar plates. Even when the treatment is carried out for 10 s, the
inhibition zone has the diameter of 1.2 cm (Fig. 4.4). As the treatment time is
increased, the area of inhibition zone also increased (Fig. 4.4). The prolonged treat-
ment of agar plates leads to the reduction of transparency of agar plates, at the area
of direct contact of effluent. This could be because of drying [8] and deformation of
agar structure due to plasma (effluent) induced chemical structural deformation
reaction. Chemically, agar is a polymer made up of subunits of the sugar galactose,
and is a component of the cell walls of several species of red algae. Algae itself is
known to undergo degradation in plasma [19]. Specifically, sugar galactose is known
to undergo photo chemical reaction [20], and argon plasma is known to produce
high energetic photons [21].
52 R. Pothiraja et al.
4
Hard agar plate
3 Soft agar plate
2
1
0 150 300 450 600
Treatment time (sec)
Fig. 4.4 Images of inhibition zone generated after plasma treatment of E. coli coated agar plates (left);
plot of inhibition zone with respect to plasma treatment time on hard and soft agar plates (right).
Studies on the decontamination of inner surface of the tube are also carried
out by coating E. coli on inner surface of the quartz tube, which is a part of our
experimental set up, from the point close to the spike till the exit point of the
effluent. In other words, E. coli is coated in the effluent region, grounded region
and the region between electrodes. Analysis of plasma treated tube shows the
logarithmic reduction of colony forming units is about 5 with the bacterial sur-
vival ratio is 0.0038%. This survival could be due the fact that some traces of
microorganisms are present in the powered electrode region, and this region is
not in contact with plasma.
Encouraged by the efficiency of decontamination of vegetative microorganism, we
have carried out efficiency studies of our plasma source for the inactivation of spore.
For this purpose, spores of B. atrophaeus are used. In order to find out the possibility
of decontamination of temperature sensitive materials, these endospores are layered
on PET polymer material. All decontamination experiments of spores are carried out
in the effluent of plasma generated in our plasma source. It is carried out in a similar
way as carried out for E. coli inhibition studies, but with the distance between the exit
point of effluent in tube and PET plate is about 1 cm. First set of experiments are
started with less number of spores (about 300) layered in a circular area within diam-
eter of a few mm on a PET plate. Analysis of plasma effluent treated plates shows that
within the treatment time of 70 s, all spores are inactivated. Hence the second set
experiments are carried out with high number of spores (about 4,700) layered in a
circular area with diameter of a few mm on PET plate. Analysis of plasma effluent
treated samples shows the complete inactivation of spore in 2 min. In other words, the
logarithmic reduction of spores is about 3.67 for the plasma effluent treatment time of
2 min (Fig. 4.5). Repetition of all the experiments shows good reproducibility.
The microorganism E. coli, which are coated inside the tube in the region between
the electrodes as well as in the region of grounded electrode, are in direct contact
with the plasma during the treatment. In this case, constituent of plasma, especially
4 Biological Decontamination Using Pulsed Filamentary Microplasma Jet 53
0
0 30 60 90 120 150
Treatment time (sec)
charged species, should have played important role in inactivating the E. coli [22]
along with photons. Since the charged species are not in direct contact with E. Coli
coated in the effluent region of the tube, their influence in inactivation of E. Coli in
this region is very less. Hence in this (effluent) region, photons produced in the
plasma and in the plasma effluent could have played important role. Also, it is con-
sidered that argon metastables played important role in inactivating the E. coli both
in active plasma region as well as in effluent region of treatment [23].
For the E. coli inhibition studies and the B. atrophaeus inactivation studies,
samples are placed in atmospheric air. These samples are treated with plasma efflu-
ent. Hence, the contribution of charged species for the inactivation will be very less
compared to the inactivation of E. coli coated tube through direct plasma. Argon
plasma, at atmospheric pressure conditions, emits photons mainly in Vacuum
Ultraviolet (VUV) and near infrared (NIR) spectral regions. The photons in NIR
region do not have enough energy to inactivate the microorganism. The VUV pho-
tons produced, as shown in the Scheme 4.1, in plasma and effluents should have
played important role in inactivation. In addition to this, oxygen in air, which is in
contact with the effluent, should have played important role as shown in the
Scheme 4.1,
The argon excimer (Ar2*) in triplet state has enough life time to reach air,
which is occupied between the sample and the exit point of plasma. This excimer
will produce VUV photons (wavelength, ~128 nm) through spontaneous emission.
This photon itself has enough energy to inactivate the microorganism, especially
E. coli, through photochemical reaction. In addition to this, this photon can dis-
sociate oxygen molecules into two oxygen atoms. This atomic oxygen can react
with microorganism through oxidation reaction and inactivate the microorgan-
isms, E. coli and B. atrophaeus [24]. The atomic oxygen can also react with
molecular oxygen to produce ozone molecule. The produced ozone molecule can
also inactivate the microorganism through oxidation reaction. By these ways,
microorganisms placed in atmospheric air could have decontaminated in the argon
54 R. Pothiraja et al.
4.4 Summary
Atmospheric pressure pulsed filamentary plasma source with argon working gas is
used for the decontamination studies. The efficiency of our plasma source for the
decontamination on inner surface of the tube as well as on objects placed in proxim-
ity of plasma effluent is studied. E. coli and spores of B. atrophaeus are used for the
decontamination studies. Inhibition studies of E. coli on agar plates are also carried
out. It shows that plasma effluent generated in our plasma source is very effective
for the inhibition of bacterial colonization. Decontamination studies of B. atropha-
eus endospores, which are layered on PET polymer material and placed in the prox-
imity of plasma effluent, show the mean logarithmic spores reduction of about 3.7
(complete inactivation) for the treatment time of 120 s. Decontamination studies of
E. coli coated on inner surface of the tube show the mean logarithmic bacterial
reduction of about 5 for the treatment time of 30 s. As a part of inactivation mecha-
nistic study, plasma is characterized using optical emission spectroscopy, current
voltage measurements, numerical simulations and microphotography. During the
characterization of plasma, nitrogen is admixed with argon and its emission is used
for determination of gas temperature, EVDF and electron density. Electron density
is in the order of 1011 cm3. The reduced electric field is in the range of 9001,700
Td. At this condition, argon ionization reaction is more probable than the argon
metastable formation reaction.
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19. Avramidis G, Stwe B, Wascher R, Bellmann M, Wieneke S, von Tiedemann A, Vil W (2010)
Fungicidal effects of an atmospheric pressure gas discharge and degradation mechanisms. Surf
CoatTechnol 205:S405S408
20. Binkley RW, Binkley WW (1972) Photochemical reactions of carbohydrates. Carbohydr Res
23:283288
21. Sobottka A, Drler L, Lenk M, Prager L, Buchmeiser MR (2010) An open argon dielectric
barrier discharge VUV-source. Plasma Processes Polym 7:650656
22. Seo YS, Mohamed A-AH, Woo KC, Lee HW, Lee JK, Kim KT (2010) Comparative studies of
atmospheric pressure plasma characteristics between He and Ar working gases for steriliza-
tion. IEEE Trans Plasma Sci 38:29542962
23. Xu G, Zhang G, Shi X, Ma Y, Wang N, Li Y (2009) Bacteria inactivation using DBD plasma
jet in atmospheric pressure argon. Plasma Sci Technol 11:8388
24. Lim J-P, Uhma HS, Li S-Z (2007) Influence of oxygen in atmospheric-pressure argon plasma
jet on sterilization of Bacillus atrophaeous spores. Phys Plasmas 14:093504
Chapter 5
The Fungal Spores Survival Under
the Low-Temperature Plasma
5.1 Introduction
H. Soukov (*)
Department of Computing and Control Engineering, Institute of Chemical Technology in Prague,
Technick 5, 166 28, Praha, Czech Republic
e-mail: hana.souskova@vscht.cz
V. Scholtz
Department of Physics and Measurements, Institute of Chemical Technology in Prague
Technick 5, 166 28 Praha, Czech Republic
J. Julk
Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in
Prague, Studnikova 7, 128 00 Praha, Czech Republic
D. Savick
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology,
Institute of Chemical Technology in Prague, Technick 5, 166 28 Praha, Czech Republic
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 57
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_5, Springer Science+Business Media B.V. 2012
58 H. Soukov et al.
which results the growth inhibition of Penicillium digitatum after two days long
corona discharge exposure, and there is an Akishevs [6] mention of the inactivation
of Aspergillus niger and Candida lipolytica on agar surface after exposure to the
plasma jet device. Soukov [7] studied the decontamination of water suspension of
fungal spores by corona discharge in detail. The possible application of plasma
sterilization may be useful e.g. for the treatment of fruits surface, preventing the
mould overgrow and bacterial putrefaction. Another possible motivation for search-
ing for the decontamination methods based on low-temperature plasma is e.g. the
subvention of The European Union for the application of low-temperature plasma
to the superficial decontamination of table eggs in connection with its plan of hen
breeding transformation, more details may be found in [8] and [9].
The mechanisms of the cells inactivation, using corona discharge, are not reliably
determined and are still open for discussion; the research in this field is advancing.
The synergic action of reactive and/or charged particles, as NOX, atomic oxygen O,
singlet oxygen 1O2, ozone O3, superoxide anion O2, hydroxyl radical OH etc.,
surrounding the cells, appears to be the most probable cause of cell wall or cell
membrane damage. To some extent, rising UV radiation can also bear certain impact.
We did not follow the presence of these particles, described in detail in, e.g. [10].
In the previous work of Scholtz et al. [11], the decontamination features of corona
discharge on the water suspension of different types of bacteria were studied (cocci,
rod shaped, gram-positive, gram-negative, extremophile). The total inactivation of
all types of bacteria by corona discharge occurred after 4 min.
The main aim of this work was to verify the devitalisation sensitivity of eukary-
otic microorganisms (fungi) to low-temperature plasma as a basic presumption of
the use of the non-thermal plasma for decontamination, and eventually sterilisation.
The other aim of the work was to compare the two types of corona discharge and to
compare their known effects on bacteria and their effects on micromycete spores.
The tolerance of four different non-pathogenic micromycetes from four different
genera was studied. The fungal species were obtained from The Department of
Biochemistry and Microbiology (DBM), Institute of Chemical Technology in
Prague. The genera Penicillium, Cladosporium and Alternaria massively occur in
the environment during summer time and can cause many allergic reactions.
1. Penicillium crustosum (DBM 4159)
2. Aspergillus oryzae (DBM 4002)
3. Cladosporium sphaerospermum (DBM 4282)
4. Alternaria sp. (DBM 4004)
The low temperature plasma was generated using the previously described sim-
ple apparatus of an open-air type [12]. The discharge burns on the point electrode,
represented by the tip of a syringe needle connected with a serial resistance of
5 The Fungal Spores Survival Under the Low-Temperature Plasma 59
Decontamination
High
Inoculation
voltage
20 M
corona Cultivation
Resistor
sample Evaluation
Fig. 5.1 The block diagram of the method used. The experimental arrangement of inactivation of
microbial suspension
20 MW to the positive or the negative pole of direct current high voltage supply.
The ground electrode was realized by the surface of water suspension of microor-
ganisms connected with an immersed platinum wire to the negative or the positive
pole of the supply. The discharge voltage was set to 9.7 kV and the distance
between the tip of the needle and water surface was adjusted to cca 3 mm to
get the discharge current of 400 mA. Due to the serial resistance in used apparatus,
the positive point-to-plane discharge burns in the regime of transient spark corona
see e.g. [13], the negative point-to-plane discharge burns in the regime of glow
corona discharge, see e.g. [14]. Some electric characteristic of used discharges are
described in [15].
All exposures were performed under laminar flow of HEPA-filtered air to prevent
the airborne contamination; the air-conditioning of the laboratory controlled the
ambient conditions.
The stock suspensions of fungal spores were prepared immediately before the
exposure to the discharge. The fungal spores were harvested using a bacteriological
loop and suspended in sterile physiological saline. Figure 5.1 shows the arrange-
ment of experiments. The 0.5 ml of the suspension was pipetted into the sterile
wells of a dot plate and exposed to the positive and the negative corona discharge
for time intervals which varied from 5 to 30 min. Following the exposure, the con-
tent of each well was diluted, spread onto the surface of YGC agar (Yeast, Glucose,
Chloramfenicol) and after 34 day cultivation at 24C, the number of the survived
colonies (CFU colony forming units) was counted. The initial concentrations of
the suspensions were also determined by the cultivation method under the hereinbe-
fore conditions.
Every result was confirmed by another two experiments. The uncertainty is pre-
sented as error bars in graphs.
Finally, to compare the growth of exposed and non-exposed spores following
experiment was done. From the stock suspension, 0.5 ml was pipetted into sterile
wells and exposed for 15 min to the positive corona discharge. Subsequently, the
exposed and non-exposed spores were cultivated under the above-mentioned condi-
tions. In regular 6-h intervals, the growing and sporulation were observed.
60 H. Soukov et al.
5.3 Results
Cladosporium sphaerospermum
1E+07
negative
1E+06 1500000
276000
concentration [cfu/ml]
250000 positive
1E+05 114000
90000
1E+04
1E+03
1E+02 100
1E+01 10
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.2 The number of survived C. sphaerospermum spores after exposure to the corona
discharge
Alternaria sp.
1E+07
1480000 940000 negative
1E+06 438000
positive
concentration [cfu/ml]
386000
1E+05 314000
52000
1E+04
7100
2000
1E+03
1E+02
22
1E+01
2
1E+00
0 5 10 15 20
exposure time [min]
Fig. 5.3 The number of survived Alternaria sp. spores after exposure to the corona discharge
5 The Fungal Spores Survival Under the Low-Temperature Plasma 61
Penicillium crustosum
1E+05
negative
12500 7600 positive
concentration [cfu/ml]
1E+04 4900
1E+01
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.4 The number of survived P. crustosum spores after exposure to the corona discharge
Aspergillus oryzae
1E+06
negative
136000
1E+05 28000 positive
concentration [cfu/ml]
25000
1E+04
1900
8000
1E+03
600
1E+02 108
1E+01
1E+00
0 5 10 15 20 25 30
exposure time [min]
Fig. 5.5 The number of survived A. oryzae spores after exposure to the corona discharge
The total inactivation of Penicillium spores occurred after 25 min, but only by the
impact of positive corona discharge. The negative corona discharge did not cause
the total inactivation even after 30 min of exposure. The positive corona discharge
has a greater effect than the negative one; it is shown in Fig. 5.4.
The fungal spores of Aspergillus oryzae appeared to be the most resistant in
comparison to the other species of fungi. The number of the survived spores fell by
three orders of magnitude, but the total inactivation did not occur even after 30 min
of exposure. Both types of corona discharge showed a comparable low effect of
devitalisation. It is shown in Fig. 5.5.
The dynamics of micromycete growth after exposure to a sublethal dose of
plasma was interesting: the surviving exposed fungal spores grew visibly slower in
62 H. Soukov et al.
Fig. 5.6 The dynamics of micromycete growth and sporulation of exposed and non-exposed
fungal spores (A. Orzyae, P. crustosum and C. sphaeerospermum)
comparison with the non-exposed ones (see Fig. 5.6). The first colonies appearance
in exposed micromycetes was delayed by 35 h for Cladosporium, by 45 h for
Aspergillus and even by 65 h with for Penicillium as compared with the non-exposed
cultures. On the other hand, the time interval between growth appearance and
sporulation was shortened substantially in cultures of the spores exposed to dis-
charge. The numbers of fungal colonies on the agar surface were in each case
approximately 50 cfu ml1, so that the above-mentioned differences cannot be attrib-
uted to different growing conditions. The micromycetes exposed (A. orzyae, P. crus-
tosum and C. sphaeerospermum) sporulated faster than the non-exposed ones. In the
previous works about bacteria and yeast [11], no similar effects were observed.
The dynamics of growth of Alternaria sp. is shown in Fig. 5.7. While all the non-
exposed spores germinated within 72 h, the exposed ones germinated even after 134 h
after inoculation. The fungus Alternaria sp. sporulated very fast and no delay in
sporulation between non-exposed and exposed but survived spores were observed.
The aim of Fig. 5.7 is to present only the time delay between the sporulation of
the exposed and unexposed spores. It would be very difficult to gain the same initial
number of CFU on the both Petri dishes; nevertheless, their numbers were very
close and do not affect the growth. From this graph, it is possible to derive the
hypothesis, that after exposure to a sublethal dose of plasma, some spores are killed,
some survive without harm and some are partially harmed and can germinate again
after revitalisation. The germination of the partially harmed spores, however, took
longer time than the germination of the intact or unexposed spores.
5 The Fungal Spores Survival Under the Low-Temperature Plasma 63
Alternaria sp.
140
120
100
80
CFU
60
40
20
0
72 134
0 20 40 60 80 100 120 140
Time of growth [hours]
5.4 Discussion
Comparing the results presented here with those obtained with bacteria, it may be
concluded that bacteria are more susceptible to the low-temperature plasma than
fungi. Using the less effective negative corona discharge, the complete inactivation
of Escherichia coli and Staphylococcus aureus was achieved after two and 4 min of
exposure, respectively [10]. The bacterial spores of Geobacillus stearothermophilus
seem to be also more susceptible to the exposure than the fungal ones [2].
The higher resistance of fungi is probably caused by the encapsulation of fungal
spores, in comparison with the unprotected cell membrane of vegetative bacteria.
The fungal cell wall is composed of a polysaccharide-based three-dimensional
network. The construction of the common central core of the cell wall is composed
mainly of branched b-1,3-glucan and chitin. Genomic as well as drug studies have
shown that the death of the fungus can result from the inhibition of the syntheses of
cell wall polysaccharide.
According to [16], the cell wall is now seen as a dynamic structure that is
continuously changing as a result of the modification of culture conditions and
environmental stresses.
Because of its essential biological role, unique biochemistry and structural
organization and the absence in mammalian cells of most of its constitutive compo-
nents, the cell wall is an attractive target for the development of new antifungal
agents. Inhibition of the fungal cell synthesis, when the cell integrity pathway is
64 H. Soukov et al.
caused by the low-temperature plasma, acting either on the envelope or the genome
of exposed spores, the reparation of which needs some time as compared with the
unexposed ones. The shorter time between the growth and sporulation of exposed
spores may be explained by the effects of stress on the spores which consider the envi-
ronment as unfavourable and by the quick sporulation try to change the living place.
5.5 Conclusion
The article presents the results of the study of fungicidal effects of negative and
positive stabilized corona discharge. The results show that the low-temperature
plasma created by corona discharge decreases the number of the survival spores
with all the fungal species tested. A 10-min-long exposure did not lead to any sig-
nificant decrease of their number, which declined approximately by one decade.
After a longer exposure, the decrease of the survival spores was more significant
and the differences among the species were perceptible.
The positive and negative corona discharges show almost the same impact on
Cladosporium sphaerospermum and Alternaria sp., whereas somewhat different
impact was observed for Penicillium crustosum and partially for Aspergillus oryzae.
We are not able to explain these differences so far.
In general, the fungal spores showed higher resistance to corona discharge expo-
sure than other microorganisms. So in possible application of low-temperature
plasma decontamination the test of fungal spores can not be omitted.
The higher effectiveness of positive corona discharge was proved with bacteria
and yeast, but this phenomenon was not confirmed with fungal spores.
The method used is in the stage of a fundamental research and its application to
larger samples or its practical use requires further experiments.
Acknowledgments This work has been supported by grants No. MSM R 6046137306, MSM
R 0021620806 and SVV-2010-26 0506.
References
6.1 Introduction
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 67
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_6, Springer Science+Business Media B.V. 2012
68 T. von Woedtke et al.
6.2 Experimental
Plasma treatment of liquids was realized using a surface dielectric barrier discharge
(surface-DBD) arrangement which was described in detail elsewhere [46]. Plasma
is generated on the surface of an electrode array. For liquid treatment in atmospheric
air conditions, this array was mounted by a special construction into the upper shell
of a Petri dish (60 mm diameter) in that way that a constant distance of 5 mm
between the high-voltage electrode surface (35 mm diameter; surface area 9.6 cm2)
and the surface of a liquid sample in the lower shell of the Petri dish (55 mm diam-
eter; surface area 23.8 cm2) can be adjusted (Fig. 6.2, left).
For liquid treatment in argon atmosphere at atmospheric pressure conditions, the
electrode array is integrated into a special housing which was constructed in that
way that it can be mounted on the lower shell of a 55-mm petri dish forming a reaction
6 Plasma-Liquid Interactions: Chemistry and Antimicrobial Effects 69
Fig. 6.2 Experimental set-up of surface dielectric barrier discharge arrangements for liquid treat-
ment in atmospheric air (left) and in argon atmosphere (right)
chamber sealing off the outside (Fig. 6.2, right). By a special gas supply channel,
this chamber can be flooded with argon gas (gas flow 0.5 slm). Corresponding to the
other experimental setup (Fig. 6.2, left), a constant distance of 5 mm between high-
voltage electrode surface and surface of liquid sample can be adjusted. As power
source a commercial Fourier synthesis pulse generator was used. In all experiments
at ambient air conditions, a pulsed sinusoidal voltage of 10 kVpeak (20 kHz) with a
0.413/1.223 s plasma-on/plasma-off time was used. For experiments in argon atmo-
sphere, a pulsed sinusoidal voltage of 3 kVpeak (40 kHz) with a 0.413/1.223 s plasma-on/
plasma-off time was used.
As test liquids, distilled water as well as sodium chloride solution (NaCl solu-
tion; NaCl 0.85%; 8.5 g NaCl per 1,000 ml water) have been used. No outgassing
procedure was performed before plasma treatment of liquids.
As test microorganism Escherichia coli NTCC 10538, kindly provided by
Institute of Hygiene and Environmental Medicine, Ernst Moritz Arndt University
Greifswald, Germany, was used. Overnight culture of E. coli was diluted using NaCl
solution, to get concentrations of 107108 colony forming units per milliliter (cfu ml1;
stock suspension).
For plasma treatment of liquids containing suspended microorganisms, 5 ml of
E. coli stock suspension were treated with the DBD plasma for different times up to
30 min. The number of viable microorganisms (cfu ml1) was estimated by the
surface spread plate count method using aliquots of serial dilutions of microorgan-
ism suspensions in NaCl solution according to the European Pharmacopoeia.
Detection limit of this procedure was 10 cfu ml1. Serial dilution of microorganism
suspensions served also as an effective procedure to neutralize the bactericidal
activity of reactive species contained in the plasma-treated liquid.
Inactivation kinetics of microorganisms is depicted in semi-logarithmic plots.
If the number of microorganisms fell below the detection limit, i.e. no viable
microorganisms have been found, for clearness these values in the graphs are set at
5 cfu ml1. Three trials (mean of n = 2 each) were done with similar concentrations
70 T. von Woedtke et al.
of E. coli suspensions (107108 cfu ml1). The kinetics of these three trials showed
the same inactivation effects for E. coli.
For plasma treatment of aqueous liquids without bacteria, 5 ml of distilled water
or sodium chloride solution (0.85%) were used.
pH measurements were done using a microprocessor pH meter pH 196
(WTW, Weilheim, Germany; measuring range/precision: 0.0014.00/ 0.01) with
a semi-micro pH-electrode (4.5 mm diameter; SENTEK P13, Sentek Ltd., UK).
For photometric measurements, UV/VIS Spectrophotometer SPECORD S 600
(analytic jena GmbH, Jena, Germany) was used. The photometer has an accuracy
of measurement of 0.5 nm in the UV range and 0.3 nm in the VIS range. The
spectral resolution is 1.21.3 nm. Nitrite and nitrate concentrations were estimated
by color forming reactions using commercially available test kits (Spectroquant,
Merck). Nitrite reacts with sulfanilic acid and N-(1-naphthyl)-ethylen diamine
hydrochloride via azo sulfanilic acid to a magenta colored azo dye whose absorp-
tion at 525 nm was measured. Nitrate reaction with 2,6-dimethylphenol gives,
after a reaction time of 10 min 4-nitro-2,6-dimethylphenol, an orange colored
product, whose absorption was measured at 340 nm. Hydrogen peroxide detection
based on the reaction of titanyl sulfate to yellow-colored peroxotitanyl sulfate,
which was detected at 405 nm.
For direct photometric analysis, total absorption spectra have been recorded from
200 up to 1,000 nm.
8,0
7,0
6,0
5,0
pH
4,0
3,0
Fig. 6.3 Change of pH in 5 ml water dependent on surface-DBD plasma treatment time in argon
atmosphere () and in atmospheric air (), and sodium chloride solution in atmospheric air () [5]
Fig. 6.4 Absorption spectra of nitrous acid (- - -), of 30 min plasma treated water (. . .) and of 30 min
plasma treated sodium chloride (NaCl) solution ( ) [5]
UV-VIS spectra of the liquids were recorded in the range between 200 and 1,000 nm
(Fig. 6.4). Absorption maximum of nitrate or nitric acid, respectively, has been
found at 305 nm [5]. In plasma-treated liquids, a distinct absorption maximum was
found at 227 nm which has been shown to be identical to the absorption maximum
of a nitrous acid (HNO2) solution at pH 2.8 which is another indication for the pre-
dominating role of HNO2 for acidification. However, at least partial occurrence of
nitrate cannot be excluded completely, particularly because nitrite partially will dis-
proportionate at higher concentrations (see Eq. 6.1). Moreover, nitrate has another
absorption maximum at 220 nm, and therefore a partial superimposition of the
227 nm peak by traces of nitrate is possible.
Nevertheless, this cannot explain the high nitrate compared to nitrite concentra-
tions in 5 ml liquid detected after 30 min surface-DBD treatment under atmospheric
air conditions as reported in our recent study [4].
The detection of nitrate was based on a color forming reaction whereby nitrate
reacts with 2,6-dimethylphenol to form the orange colored 4-nitro-2,6-dimethylphenol
within a reaction time of 10 min:
(6.2)
with the indicator substance. But, as mentioned already, under acidic conditions a
disproportionation of NO2 into NO3 takes place (see Eq. 6.1).
If NO3 as the product of this reaction is consumed by an indicator, the equilib-
rium of this reaction is moving to this product with the result that by and by the
complete nitrite is transformed into nitrate resulting in high nitrate concentrations as
detected. Because nitrite and nitrate have been measured in different aliquots of the
plasma-treated liquid, this transformation of nitrite forced by the analytics of nitrate
was not noticed. Only with direct UV-VIS spectrometry of the liquids (see Fig. 6.4)
without color forming and analyte consuming reactions this artifact can be
avoided.
Moreover, from a chemical point of view, also a direct reaction of the NO2 radi-
cal with the phenol ring of 2,6-dimethylphenol is conceivable:
(6.3)
109
107
(cfu . ml -1) 106
105
104 argon
103
ambient air
102
101
detection limit
100
0 5 10 15 20 25 30
plasma treatment time [min]
Fig. 6.5 Inactivation kinetics of 5 ml E. coli suspensions in non-buffered NaCl solution dependent
on surface-DBD plasma treatment time in argon () an ambient air atmosphere ()
to 6 mg l1 in 5 ml liquid within 30 min, only (data not shown). In the same liquid
volume, a H2O2 equivalent concentration of 18 mg l1 was measured after 30 min
surface-DBD treatment in atmospheric air [4]. But, neither NO3 nor NO2 related
concentrations were detectable if the liquid was surface-DBD treated in an argon
atmosphere. The last fact is not surprising because of the lack of air in this test
system. However, mechanisms of H2O2 generation as well as acidification have to
be investigated in more detail. One possible explanation is plasma-induced water
dissociation which has been discussed as a result of plasma generation directly in
water [12].
According to the results presented here, reactive species containing both nitro-
gen and oxygen seem to be necessary for antimicrobial effectivity because such
species could not be detected after plasma treatment in argon atmosphere. Nitrate or
nitrous acid, respectively, has been identified to play a key role in acidification.
There are some reports in literature about more or less distinct antimicrobial activity
of nitrite under acidic conditions whereas it is hypothesized that not nitrite itself but
other reactive species arising from further reactions are responsible for the antimi-
crobial activity [1315].
In this context it has to be taken into consideration that plasma-generation in
argon has been realized using lower energy input compared to air (3 kV vs. 10 kV
ignition voltage). This was inevitable because, according to the Paschen curves
higher ignition voltage is needed to generate plasma in air compared to argon. On
the other hand, the use of 10 kV ignition voltage in argon atmosphere was not pos-
sible in this experimental setup because of intensive spark generation. However,
because the surface-DBD plasma was not in direct contact with the bacteria contain-
ing liquid, from our point of view bactericidal effects of different electric fields
should be unlikely.
6 Plasma-Liquid Interactions: Chemistry and Antimicrobial Effects 75
Acknowledgements This study was realized within the joint research project Campus
PlasmaMed supported by the German Federal Ministry of Education and Research (grants
no. 13 N9779 and 13 N11188).
6 Plasma-Liquid Interactions: Chemistry and Antimicrobial Effects 77
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Chapter 7
Damages of Biological Components in Bacteria
and Bacteriophages Exposed to Atmospheric
Non-thermal Plasma
7.1 Introduction
Emergence of the next pandemic influenza, avian flu (H5N1) or swine flu (H1N1),
and drug resistant bacteria are of concern for public health. Non-thermal atmospheric
pressure plasma is effective for dealing with such menaces caused by bio-particles
(BPs) because, in principle, it can decontaminate both the surface of materials [1, 2]
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 79
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_7, Springer Science+Business Media B.V. 2012
80 A. Mizuno and H. Yasuda
and room air [3, 4] at low gas temperature without using bactericides. Though the
study of plasma decontamination is expanding [59], the mechanism of inactivation
of BPs are still to be studied. In this study, we have evaluated the damages to E. coli,
B. subtilis exposed to dielectric barrier discharges [10]. GFP (green fluorescent
protein) in E. coli can be damaged quickly by the exposure to the DBD (dielectric
barrier discharge), and the intensity of GFP and survivability shows similar trend
against the exposure time. Turbidity of B. subtilis solution decreases quickly with
the exposure to the DBD. We have also developed a biological assay to evaluate
DNA-specific damage in bacteriophages treated with DBD. It was found that l
phage DNA was strong against the plasma application in comparison with coat
proteins. Phages having single stranded DNA, however, can be damaged as sensi-
tive as its coat protein.
7.2 Experimental
BPs are commonly existing around us, and electrostatic precipitation can collect
suspended BPs in air with high efficiency and low pressure drop. In the meantime,
corona discharges in ESPs generate radicals and other oxidative particles. Those are
effective to destroy BPs. Previous experimental work indicated effective destruction
of yeast cell on the collection electrode of a corona discharge system [11]. We have
tested the destruction effect of a corona discharge generated in the needle-plate
system. Bacteriophage MS2 can also be destroyed, with improved efficiency when
the phages are in wet condition.
Film or culture media can be used as the collecting electrode. Figure 7.1a illus-
trates the ESP used in this study. The needle electrode with DC high voltage was
placed above the YPD (Yeast extract Peptone Dextrose) medium, and corona dis-
charge was generated between them. Distance between the needle electrode and
YPD medium was 40 mm. The ESP was placed in a box and a fan was set at the top
of the box to introduce the air from the upper side and the BPs are collected on the
YPD medium. Different voltage was tested (030 kV) on each fresh YPD plate.
Collected samples were incubated at 30C for 48 h for the colony forming.
Figure 7.1b shows the effect of ESP on the BPs collection. Particles in room air
were collected on YPD medium for 1 min. Positive and negative voltages were exam-
ined in this experiment. The result shows that, for both polarities, the number of
collected bacteria increased with applied voltage. However, the positive corona dis-
charge shows the higher efficiency than the negative corona. The highest collection
efficiency was observed with applied voltage of 25 kV. This efficiency suggesting
that collection of bacteria was enhanced by about 50 times compared with non-
applied discharge BPs amount. In addition, influence of applied voltage and distance
7 Damages of Biological Components in Bacteria 81
Fig. 7.1 Collection of suspended bio-particles in air using electrostatic precipitation. (a) An elec-
trostatic precipitator for collecting bio-particles in air. (b) Number of colony due to collected
bio-particles using the ESP
Fig. 7.2 Radial distribution of the colonies collected by electrostatic precipitation. X-axis: relative
density of colonies. Y-axis: distance from the center (just under the needle electrode). (a) Positive
corona (8 and 13 kV). (b) Negative corona (8 and 13 kV)
of electrodes on collection profiles of BPs was noticed, as shown in Fig. 7.2. In this
case, the distance between the needle electrode and the surface of YDP media was
10 mm. This small distance was selected to measure the effect of radial distance
more clearly. Relative density of the colonies was measured for comparison. This
was calculated by measuring the colonies in a concentric circle of 9 mm width, and
divided by total number of colonies. Close to the center of the plate, just under the
high voltage electrode, the relative colony density increased. However, above a cer-
tain applied voltage, the relative colony density at the center decreased. The total
number of colonies (Fig. 7.1b) also decreased with very high voltage. At 30 kV, the
number of collected bacteria decreased. Due to ionization of corona discharge, radi-
cals such as O, OH are generated, and from these short-lived radicals, long lived
oxidative species are generated. From mass spectroscopy, it is known that corona
discharge generates stable neutral O3 molecules, and ions such as O3(H2O)
82 A. Mizuno and H. Yasuda
Figure 7.4 illustrates the dielectric barrier discharge (DBD) reactor used in this
study. Stainless steel mesh (diameter: 50 mmj, 20 mesh (separation between adja-
cent wire of the mesh = 1/20 in.)) and aluminum plate were used as high voltage and
GND electrodes respectively. Teflon sheet (2 mm-thick) was set on the high voltage
electrode as a dielectric barrier. A high voltage AC power supply (Kasuga-denki
AGF-010) was used to generate the uniform filamentous streamers in the
7 Damages of Biological Components in Bacteria 83
atmospheric air gap (3 mm). All of the discharge experiments in this report were
done in a fixed electrical condition except operating time. The peak to peak voltage,
the frequency of the applied voltage and the input power were 40kVp-p, 2 kHz and
88 W, respectively. A piece of polyethylene terephthalate (PET) film (90 mm 30 mm,
0.1 mm thickness) was soaked in 0.1% gelatin for 5 min and air dried for 24 h under
UV light. 20 ml of sample solution was spotted and widely spread to 34 mm2 on the
PET film and immediately applied the atmospheric plasma for intended time. After
the DBD application, the sample solution was recovered into a microtube with addi-
tional washing by 100 ml of distilled water on the surface of the PET film. The
recovered samples were rendered for experiments of survival estimation, fluores-
cence measurement, protein analysis, DNA analysis and microscopic observation.
Green Fluorescent Protein, GFP, coding plasmid pGLO (5.4 kb in size) was
purchased from Bio-Rad Co. Inc. and transfected to E.coli MV1184. The transformants,
E.coli MV1184(pGLO), were propagated by shaking at 37C for over night in 20 ml
of LB medium supplemented with 5 mM L-arabinose which induces and accumu-
lates GFP in the cells. The cells were harvested by centrifugation at 5,000 g for
5 min, washed and finally suspended in 3 ml of 100 mM Tris HCl(pH8.0).
Bacteriophage lambda (lCI857Sam7) was induced from l lisogen, E.coli M65.
The E.coli cells were inoculated to 200 ml of LB medium in a 1 L flask and shaken
at 32C. When OD650 (optical density at 650 nm wavelength) reached to 0.5, cul-
tivation temperature was quickly shifted to 42C and shaken for 20 min and then
shaken for 3 h at 40C. The cells were harvested by centrifugation at 8,000 g for
5 min and resuspended with 10 ml of SM buffer. The cells were lysed by adding
0.1 ml of chloroform and 10 ml of 2 mg/ml of pancreatic DNase and gentle shaking
at 37C for 20 min. The cell lysate was centrifuged at 10,000 g for 10 min and
recovered the supernatant. Further purification of the lphage in the lysate by step-
wise CsCl density gradient centrifugation and CsCl equilibrium density gradient
centrifugation was performed according to the literature protocol [13] except that
the phage was finally dialyzed against 100 mM Tris HCl(pH8.0), 1 mM MgCl2.
Purified DNA from E.coli MV1184 (pGLO) was obtained by serial extraction of
the cells with phenol, phenol-chloroform, and chloroform as described in [6]. After
precipitation with ethanol, the DNA was dissolved in 100 mM Tris HCl(pH8.0),
1 mM EDTA.
GFP was purified from E.coli MV1184(pGLO) following the protocols of cell
lysis and affinity column chromatography using hydrophobic resin from Bio-Rad
Co. Inc. The DBD treated cells were serially diluted with SM buffer and plated on
LB plates. After incubation at 37C for 30 h, colonies on the plates were counted.
Titration of the DBD treated lphage was done as follows. As indicator cells,
E.coli Y-mel was cultivated in LB medium at 32C for over night. 0.1 ml of the seri-
ally diluted lphage solution was mixed with 0.1 ml of the indicator cell suspension
and kept at 37C for 10 min. Three milliliter of LB soft agar kept at 45C was added
to the mixture of infection and poured on a LB plate. Phage plaques on the plates
were counted after incubation at 40C for 30 h.
All of the survival measurement experiments were duplicated independently and
the mean value was adopted.
84 A. Mizuno and H. Yasuda
Figure 7.5 also indicates the fluorescent intensity of GFP in E.coli MV1184(pGLO)
cells after time-lapse treatment with DBD. The intensity decreased with increasing
the time of DBD treatment, and complete inactivation of the GFP function was seen
at 40 s of the treatment. The exhibited results recall some correlation to the cell
survival. Pouring alkali solution to the bleached samples did not improve their fluo-
rescence though normal (non-discharged) GFP inactivated in acid solution exhib-
ited full recovery of the fluorescence by neutralization with alkali. It was suggested
that the bleaching of GFP by DBD treatment seemed to be caused by its irreversible
denaturation or chemical modification or degradation.
Figure 7.6 shows the SDS (sodium dodecyl sulfate) polyacrylamide gel electro-
phoresis of E.coli MV1184(pGLO) subjected to the atmospheric DBD. Cells were
lysed and fractionated in a 14% gel before staining with Coomassie Brilliant Blue
(CBB). Lane M is a protein standards marker, lane 1 is purified GFP, lane 2 is non-
induced E.coli MV1184 (pGLO), Lanes 38 represent the cells treated with DBD
for 0, 5, 10, 20, 30, 40 s, respectively. The arrow indicates GFP.
The thick band (indicated by the arrow) of the lane 38 corresponds to GFP. GFP
and many other intrinsic proteins of E.coli were not degraded significantly. Even in
7 Damages of Biological Components in Bacteria 85
Fig. 7.5 Survival rate of the E.coli after the DBD treatment. DBD dielectric barrier discharge,
CFU colony forming unit
Fig. 7.6 SDS polyacrylamide gel electrophoresis of E.coli MV1184(pGLO) subjected to the
atmospheric DBD. Cells were lysed and fractionated in a 14% gel before staining with coomassie
brilliant blue (CBB). Lane M is a protein standards marker. Lane 1 is purified GFP. Lane 2 is non-
induced E.coli MV1184 (pGLO). Lanes 38 represent the cells treated with DBD for 0, 5, 10, 20,
30, 40 s, respectively. The arrow indicates GFP
the 40 s discharge treated sample (lane 8), considerable amount of full length GFP
was remaining though its fluorescence was completely lost (Fig. 7.5). These results
suggest that the cause of the GFP bleaching by DBD treatment was irreversible
denaturation of its tertiary structure or chemical modification by oxidation or reduc-
tion but not the degradation of the peptide bonds.
Figure 7.7a and b shows the analysis of DNA from the DBD treated cells by
agarose gel electrophoresis. The chromosome DNA was slightly cut by sharing
force of mixing before separation with electrophoresis. In Fig. 7.7a, the chromo-
somal DNA was separated into two different positions on the 0.3% agarose gel. One
stayed near the sample well which did not enter the gel matrix and the other
migrated to the position slightly larger than the l phage DNA (48.5 kb). The pattern
of the chromosomal DNA bands did not change until 30 s of the DBD treatment.
86 A. Mizuno and H. Yasuda
Fig. 7.7 Agarose gel electrophoresis of the cellular DNA from E.coli MV1184 (pGLO) treated by
the DBD. (a) DNA was extracted and separated in 0.3% gel before staining with ethidium bromide.
Lane M1 is hind digests of lDNA, Lane M2 is monomeric lDNA. Lanes 16 represent the DNA
from the cells treated with DBD for 0, 5, 10, 20, 30, and 40 s, respectively. Arrows indicate chro-
mosome DNA. (b) Plasmid DNA fractionated in 0.8% gel. Lane M represents the purified pGLO
DNA. Lanes 16 represent the plasmid DNA fraction from the cells treated with DBD for 0, 5, 10,
20, 30, and 40 s, respectively. sc, oc and l represent super-coiled, open circler, and linear form,
respectively. (c) Plasmid DNA fractionated in 0.8% gel from the purified DNA subjected to DBD.
Lane M represents the purified pGLO DNA. Lanes 16 represent the naked DNA samples treated
with DBD for 0, 5, 10, 20, 30, and 40 s, respectively
Some degradation was seen in the 40 s treated sample which seems to be caused by
increased acidity of the sample because DNA is labile to acid. It is concluded that
the chromosomal DNA did not degraded remarkably during inactivation of E.coli
cells by DBD treatment. Bright materials at the bottom of the gel are ribosomal
RNA. Similarly to the DNA, influence of the DBD treatment to the ribosomal RNA
was not detected. Plasmid DNA is a good reporter molecule about DNA degradation
because only a nick introduction changes its topological structure from super-coiled
to relaxed form. Figure 7.7b shows the plasmid DNA (pGLO) fraction separated
from the DBD treated cells on 0.8% agarose gel electrophoresis. The bands at lower,
upper and middle positions correspond to super-coiled, relaxed (open circular) and
linear form of the plasmid molecules, respectively. The appearance of three types of
molecular form represents some degradation of the plasmid DNA has occurred, but
it seems to be caused by cellular intrinsic DNase during recovery of the samples
because non-discharged sample (0 s sample) also contained the three types of the
DNA (Fig. 7.7b, lane l control). The pattern of the plasmid DNA bands did not
change until 30 s of the DBD treatment which means that the nick caused by the
discharge was introduced scarcely until 30 s of the DBD treatment to the cells
(Fig. 7.7b, lane 25). The degradation of the plasmid seen in lane 6 may be caused
by acidic effect. Figure 7.7c shows the plasmid DNA fractions from the DBD
7 Damages of Biological Components in Bacteria 87
Fig. 7.8 E.coli cells which produce GFP with and without the exposure to the DBD. (a)(c) fluo-
rescent of GFP (d)(f) fluorescent of DNA by addingYOYO-1
applied purified E.coli DNA solution (naked DNA). With the lapse of discharged
time, super-coiled molecules have decreased and relaxed molecules have increased
(Fig. 7.7c, lane 26). The rational change of the isomeric form of the plasmid DNA
indicates the nick introducing activity was present in the DBD treatment to the
naked DNA. The inertness of the DBD for the DNA inside the bacterial cells
(Fig. 7.7b) may be caused by the protective effect of gram negative bacterial cell
envelope (inner membrane, outer membrane, periplasm and cell wall). In any case,
DNA destroying activity of the DBD treatment was very small, though it should be
reminded that subtle changes in DNA may largely affect to cell viability.
Images of E. coli cells which produce GFP were shown in Fig. 7.8 with and with-
out the exposure to the DBD. Originally the fluorescent was very bright, and the cells
were clearly observed (Fig. 7.8a). With the exposure to DBD, the cells gradually
changed into faint images because of the bleaching of GFP (Fig. 7.8b, and c). The
observation agrees well to the results of fluorescent spectroscopy. Figure 7.8d, and e
show images of the DBD applied cells stained with YOYO-1(1,1-((4,4,7,7-
Tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-
oxazole)-2-methylidene)quinolinium tetraiodide), a fluorescent dye binds specifically
to double stranded DNA. The restored blight images of the 30 s DBD treated cells
stained with YOYO-1 (Fig. 7.8d) means that considerable amount of chromosomal
DNA was remaining inside the cell without extreme degradation; this is consistent
with the results from DNA analysis by electrophoresis. The cells over-sterilized with
80 s discharge showed tight aggregation. Those cell wall might have been destroyed
and interconnected with each other (Fig. 7.8e). Figure 7.8f shows the YOYO-1
stained host MV1184 cells which do not produce GFP. Only a fraction of the cells
88 A. Mizuno and H. Yasuda
were strongly stained and other major fractions were stained weakly. The bright cells
seem to be dead ones because YOYO-1 does not intrude easily inside the living
healthy cells. Increasing of the rate of brightly stained cells by DBD treatment
(Fig. 7.8d) strongly suggests that cell wall and cell membrane are damaged and
destroyed, at least locally or in a small manner, during the sterilization.
Damages on the cell membrane have been thought to be essential for bacterial ster-
ilization by low temperature plasma. Bacteriophages usually do not have membrane
and consist of only proteins and nucleic acids. It is interesting to investigate the
effect of the plasma to membrane free bacteriophages.
Figure 7.9 shows the inactivation profile of the lphage after time-lapse treatment
with the DBD. Number of infectious phage decreased quickly and complete inacti-
vation was achieved by 30 s discharge treatment. The rate of inactivation was higher
than that of E. coli in the same electrical conditions of DBD. The profile exhibited
a characteristic of a single slope curve and the D-value was about 5 s.
Figure 7.10a shows the analysis of proteins from the DBD treated lphages by
SDS polyacrylamide gel electrophoresis. lphage has about 20 genes of coat proteins,
but only two major proteins were detectable on the gel (Figure 7.10a lane 14). These
proteins degraded rapidly comparing to the E. coli cellular proteins and could not
detect in the 30 s discharged sample. Though the degradation rate of the proteins was
slow comparing to the inactivation rate of the phage, the time of the disappearance of
protein and the completion of the inactivation coincided. The exposure of phage to
the solution of outer environment might lead to the high sensitivity of the protein to
the DBD action. Inactivation of the phage proteins involved in binding to the cell
surface receptor may contribute largely to the decrease of infectious phage.
7 Damages of Biological Components in Bacteria 89
Fig. 7.10 Analysis of protein and DNA from bacteriophage l subjected to the DBD. (a) SDS polyacryl-
amide gel electrophoresis of bacteriophage l subjected to the atmospheric DBD. Bacteriophagel was
lysed and fractionated in a 14% gel before staining with CBB. Lane M is a protein standards marker. Lane
0 represents a large amount of purified l phage. Lanes 16 represent the proteins from the phages treated
with DBD for 0, 5, 10, 20, 30, 40 s. (b) A 0.3% agarose gel electrophoresis of DNA from bacteriophage
l subjected to the atmospheric DBD. Lane M1 is monomeric DNA. Lane M2 is hind digests of lDNA.
Lanes 16 represent the DNA from phages treated with DBD for 0, 5, 10, 20, 30, 40 s
Fig. 7.11 The method to estimate the DNA-specific damage in plasma applied l phages
Figure 7.10b shows the analysis of DNA from the DBD treated l phages by 0.3%
agarose gel electrophoresis. The phage DNA degraded faster comparing to the
E. coli cellular DNA and could not detect in the 40 s discharged sample, but the
degradation was slow when compared to the inactivation rate of the phage. Here
also, the protective function of the cellular membrane from the attack of discharge
to the DNA was suggested.
Figure 7.11 illustrates the method to estimate the DNA-specific damage in plasma
applied lphages. Putative damage is introduced in both protein and DNA of the
90 A. Mizuno and H. Yasuda
Fig. 7.12 Plaque forming unit of lphage and M13 phage subjected to the DBD. After the DBD
exposure, the double stranded DNA oflphages were extracted and re-packaged, or the single
stranded DNA of M13 phages were extracted and transfected to E. coli. (a) PFU curves of plasma
treated l phages, and the re-packaged phages. (b) PFU of the DBD treated M13 phages and
Relative PFU curves obtained from the transfection of recovered single stranded DNA after the
exposure to the DBD
plasma treated phages (Phage A). DNA is extracted from Phage A and packaged
in vitro to form newly packaged phages (Phage B). Phage B does not have protein
damage and carries only DNA damage originated from Phage A. Therefore, all of the
inactivation factors in Phage B originate DNA damage brought from Phage A.
Because re-packaging procedure of lphage usually decreases the efficiency of infec-
tion, absolute number of active phages (phage titer) in Phage A and Phage B can not
be compared directly. Comparison of the normalized survival curves from Phage A
and Phage B enables to evaluate the DNA damage and the protein damage.
Figure 7.12a shows the relative PFU (plaque forming unit) curves obtained from
the re-packaged lphages and the plasma treated phages. These PFU curves were
normalized with each control sample (0 s samples) to give the PFU value of 100. The
profile exhibited the characteristic of a single slope curve until 20 s discharge and
the D value was about 25 s. Large D value of the re-packaged phages means very
slow decrease of infective phages until 20 s. The results indicate that the DNA dam-
age introduced by plasma was very small and did not accumulate prominently with
increase of the discharge time. It seems that all of the initial damage for inactivation
in phages introduced by plasma was protein damage, and DNA damage was marked
on the phages already inactivated by protein damage. So, it is concluded that inacti-
vation of lphage by atmospheric DBD was attributed to the damage of coat pro-
teins. The damage of lDNA was negligible in the early stage of the inactivation.
The damage responsible for phage inactivation can be recognized only when the
assay of phage viability was carried out. Therefore, the damage for inactivation may
not be directly correlated to the amount of the molecular damage. For example,
molecular damage introduced in the binding protein which interacts with phage
receptors on E. coli cell surface may largely affect to the inactivation. Moreover, it
should be counted that cells have a highly developed DNA repair system. Molecular
damage of DNA might have been repaired effectively than that of proteins.
7 Damages of Biological Components in Bacteria 91
The bio-assay of DNA damage has enough reproducibility when the strains of
phage and host cell were fixed. This assay can be applied to not only plasma inactiva-
tion but also any type of inactivation step of bacteriophages. Application of the DNA
damage assay to other plasma sources or different sets of bacteriophages and host
cells may bring valuable insights into the mechanism of plasma inactivation.
For comparison, damages to M13 phages were evaluated using the same proce-
dure. After the exposure to DBD, the DNAs (single stranded) were extracted, and
were transfected to E. coli to measure the infection rate. The result as shown in
Fig. 7.12b indicated that the infection ability did not recover. The comparison shows
that double-stranded DNA is stronger against the exposure to DBD, as the DNA
repairment system of the host cell works if the damage remains on one strand.
7.4 Conclusion
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Chapter 8
Investigations of Bacterial Inactivation
and DNA Fragmentation Induced by Flowing
Humid Argon Post-discharge
8.1 Introduction
Non-thermal plasma technologies have been heavily investigated during the last
decade for biomedical applications including surface decontamination of thermally
sensitive materials [13]. Several techniques were studied with different excitation
sources operating in different gases at low pressure [4, 5] and atmospheric pressure
[59]. The dielectric barrier discharge (DBD) process under investigation operates at
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 93
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_8, Springer Science+Business Media B.V. 2012
94 E. Odic et al.
atmospheric pressure in humid argon. Argon is preferred to air as a feed gas in order
to avoid the formation of ozone and nitrogen oxides (and nitric acid if water is present
[9]) which may damage the treated surface. Furthermore, water dissociation,
producing highly oxidative species, is much more efficient in the presence of argon
in the discharge. The contaminated surfaces to be treated are exposed to a non
emissive flowing post-discharge, i.e. to the discharge products, without direct contact
of the plasma (here in a filamentary regime) with the surface. Such a remote exposure
mode was chosen in order to obtain a homogeneous surface treatment and to minimize
surface material degradation. During the interaction of the discharge with a microor-
ganism, DNA can be released from cells [10] and damaged [11]. In the present work,
inactivation of a model planktonic microorganism and degradation of DNA mole-
cules in solution were investigated in separate experiments in correlation with the
nature of the active species transported by the flowing post-discharge.
For most of the experiments, the DBD reactor (reactor 1 in Fig. 8.1) consisted of a
stainless steel rod (2 mm diameter) centered in a dielectric Pyrex tube (6 and 3 mm
external and internal diameters respectively) externally covered with a 24 mm length
stainless steel mesh (allowing optical emission spectroscopy) connected to ground.
The inner electrode was connected to an AC (530 kHz) high voltage power supply
(0.510 W discharge input power). The DBD reactor was fed with humid argon (at
room temperature, 0.52 L/min). The outlet of the discharge tube was connected to
the treatment vessel in which biological samples were exposed to the flowing post-
discharge. More precisely, biological samples were placed 5 mm from the tube
outlet, 15 mm from the discharge zone itself.
In a second reactor geometry (reactor 2, see Fig. 8.1), the electrode system consisted
of two circular plane electrodes (brass) separated by an alumina (Al2O3) dielectric disc
(2 mm thickness and 60 mm diameter). The same high voltage power supply as for reac-
tor 1 was used; the high voltage electrode and the grounded electrode were 30 and
26 mm diameter respectively. Due to the size difference between the metallic electrodes,
the higher electric field region is located along the triple line (gas/metal/dielectric)
corresponding to the circumference of the smaller diameter plane electrode (grounded
electrode). Surface discharges then propagate on the alumina surface on one side of the
ceramic disc. A Pyrex dish, onto which the biological samples were deposited, was
placed facing this surface discharge (i.e. the lower side of the Al2O3 disc in Fig. 8.1). The
distance between the biological samples and the surface discharge was fixed to 30 mm.
Arcal 1 grade argon from Air Liquide was used (O2 < 0.5 ppm, N2 < 1 ppm, H2O
< 1 ppm) in all experiments. The gas flow was controlled using Brooks Sho-rate rotameters.
8 Investigations of Bacterial Inactivation and DNA Fragmentation 95
Feed gas
HV power HV power
Feed gas
supply supply
Pyrex tube
Al2O3disc
NTP source: DBD
volume discharge NTP source: DBD
surface discharge
Water vapor was added to argon by bubbling through distilled water in a gas sparging
bottle at room temperature. Water vapor content was adjusted by mixing dry argon and
water saturated (at room temperature) argon, while keeping total flow rate constant.
Hydrogen peroxide, oxygen and hydrogen are end products of water dissociation.
Gas phase chemical measurements of hydrogen and oxygen were simultaneously
made using an Agilent 6890A gas chromatograph (GC) with a Restek 100/120
Shincarbon-ST 2 m 1 mm column and a thermal conductivity detector. Argon was
used as the GC carrier gas. Hydrogen peroxide was collected by absorption in dis-
tilled water (Henrys Law constant = 105 M/atm), in the form of water droplets or
water film, submitted to the plasma post-discharge for various exposure times.
Using absorption spectroscopy (Perkin Elmer Lambda 15 spectrometer) of hydro-
gen peroxide-vanadate complex at ~430 nm, liquid phase measurement of hydrogen
peroxide concentration was made. It should be noted that the ppm units for hydro-
gen and oxygen are in the gas phase (volume/volume), and should not be confused
with the ppm units for hydrogen peroxide in liquid phase (weight/weight).
The bacteria strain used to evaluate the surface decontamination activity of the
flowing humid argon post-discharge was Escherichia coli (Gram-negative bacterium,
96 E. Odic et al.
DNA solutions in the form of 1020 mL droplets were spotted onto sterile glass
slides and were then exposed to the plasma treatment in the same way as had the
bacteria samples. Genomic DNA (100 mg/mL denatured salmon sperm DNA) and
plasmid (50 mg/mL PET9SnI: 4,285 bp circular double stranded DNA) were used
as model compounds. After treatment, according to the experimental conditions, the
sample could be dried or not. DNA was collected by four successive rinses with
10 mL distilled water droplets. The resulting solution was then analyzed. Agarose
gel (Tris Acetate EDTA TAE buffer solution pH8) electrophoresis was used to sepa-
rate DNA molecules according to their molecular weight and 3-D structure. Before
deposition of the DNA solution (20 mL) in the wells, 2 mL of negatively charged
loading buffer solution were added. After migration, the agarose gel was immerged
for 15 min in an ethidium bromide (EtBr intercalated into DNA) solution (0.5 mg/
mL) for DNA migration band visualization by UV light.
The major stable products of water dissociation were measured in the flowing post-
discharge (O2 and H2 concentration in the discharge effluent using GC-TCD technique)
8 Investigations of Bacterial Inactivation and DNA Fragmentation 97
a.u.
9
8
7
6
5
4
3
2
1
0
200 300 400 500 600 700 800 900
wavelength (nm)
Fig. 8.2 Discharge emission spectrum for 50% RH at room temperature. Light collected by an optical
fiber, through the Pyrex wall and counter electrode (stainless steel mesh). Reactor 1: 3 W/2 L/min
of reactor 1 while optical emission spectroscopy was used in order to evaluate the OH
emission (in the discharge since the post-discharge is non emissive). Flow rate and
input power were maintained constant, 2 L/min and 3 W respectively, when the water
vapor content in argon was decreased at room temperature from 80% RH down to
near dry conditions (the elimination of trace water could not be obtained without
heating the device). An example of obtained emission spectra (OH* at 308 nm) is
presented in Fig. 8.2 and further results in Fig. 8.3.
The first major result found was that the smaller the humidity, the greater the OH
emission, down to a concentration range of 150800 ppm H2O, after which the
emission began to decrease (although never reaching zero due to trace water; the
lowest water vapor concentration presented in Fig. 8.3 corresponds to about 30 ppm).
The rise in OH emission with water content for very low humidity is due to a rising
production rate of total OH, while the decrease for even higher levels of water con-
tent is due to increased quenching of excited OH by water vapor, leading to greater
proportion of OH existing in the ground state.
This quenching reaction of OH*(A2S+) by water molecule can be found in litera-
ture [12] where the following mechanism was proposed:
Ar* ( p ) + H O Ar ( p ) + OH (A ) + H
4
2
3 * 2 +
(8.1)
( ) (
OH* A 2 + + H 2 O OH X 2 + H 2 O ) (8.2)
The range of 150800 ppm water content is close to the humidity for obtaining
the maximum emission by excited OH found in [12] (~200 ppm). Of course, emis-
sion of excited OH is not a direct measure of total hydroxyl radical concentration
nor of its evolution with water vapor content. The production of hydrogen and
98 E. Odic et al.
250 1
ppm H2
ppm O2
150 0,6
H2, O2 ppm
100 0,4
50 0,2
0 0
0 5000 10000 15000 20000 25000 30000
ppm H2O
Fig. 8.3 Maximum normalized OH emission (308 nm) in the discharge zone, and hydrogen
oxygen concentration measured in the discharge effluent vs. water vapor content in argon. Reactor
1: 3 W/2 L/min
oxygen rises with humidity from dry conditions to ~2,00010,000 ppm H2O and
then slowly decreases. As mentioned above, direct measurement of total OH con-
centration has not been made but the production of hydrogen peroxide was studied.
Hydrogen peroxide formation results from:
OH + OH + Ar H 2 O2 + Ar (8.3)
in H2 production seen in Fig. 8.3. This implies that the observed decay in H2
production may come from increased quenching of energetic electrons and argon
metastable by increasing water content.
The same humid argon discharge effluent was investigated for the purpose of surface
decontamination. Glass slides contaminated with E.coli (~106 bacteria suspended in
a 10 mL water droplet) were exposed to the discharge effluent in comparable condi-
tions (2.6 W 2 L/min 95% RH in argon) and for increasing treatment time. In
order to estimate the loss in decontamination efficiency which should be caused by
longer transfer time of the activated species (short lived species such as OH and HO2
radicals and excited Ar* atoms) from the plasma source, a 525 mm long PFA tube
(6 mm ID) could be inserted between the outlet of the DBD tube and the biological
samples. Figure 8.4 shows the results (survival curve, i.e. number of survivors vs.
exposure time) obtained in both conditions: samples exposed to the flowing post-
discharge (1) directly at the DBD tube outlet (15 mm from the discharge source) and
(2) at the outlet of the PFA tube (540 mm from the discharge source). A reduction of
six orders of magnitude of E. coli population was achieved within 15 and 40 min for
the short distance and long distance experiments, respectively.
N (E. coli )
1,E+07
controls
1,E+06
1,E+05
1,E+03
15 mm from source
1,E+02
1,E+01
1,E+00
1,E-01
0 5 10 15 20 25 30 35 40 45
Treatment time (min)
Fig. 8.4 E. coli survivors vs. exposure duration to DBD flowing post-discharge. Reactor 1: 2.6 W
2 L/min humid argon
100 E. Odic et al.
70 20
H2O2 15 mm from plasma source
18
60 H2O2 540 mm from plasma source
16
H2O2 in aqueous phase ppm
20 6
4
10
2
0 0
0 2 4 6 8 10
Treatment time (min)
Fig. 8.5 Evolution of hydrogen peroxide concentration in a 3 mL distilled water film submitted
to a humid argon flowing post-discharge (reactor 1: 2.6 W 2 L/min humid argon); grey diamonds
at 10 min correspond to the hydrogen peroxide concentration measured in a 3 mL distilled water
film submitted to a flowing hydrogen peroxide/argon gas mixture (2 L/min) through reactor 1
(discharge off)
in Fig. 8.4 at 10 min) are very close to those observed with the 10 min plasma
treatment with exposure at the outlet of the long PFA tube (0.38 log reduction),
but far inferior to those observed with the 10 min plasma treatment with direct
exposure at the DBD tube outlet (4.3 log reduction). It can then be assumed that
the surface decontamination effect observed (1) for a long distance from the
source is due to hydrogen peroxide and (2) for a short distance from the source
could be due to the combination of hydrogen peroxide and short-lived species
produced by the discharge. In the latter case, the action of hydrogen peroxide can-
not be neglected since, as shown in Fig. 8.5, its concentration in the water phase
is increasing faster for short distance from the source, even if gas phase produc-
tion of hydrogen peroxide remains constant, because of water evaporation. This
phenomenon will be drastically increased in the case of water droplets (in which
bacteria are suspended) as opposed to 3 mL water films. A second remark is that
at 2 L/min, the transit time of active species from the discharge to the surface
(15 mm from the source) was estimated to 25 ms. The interaction of short lived
species can thus not be neglected. This point was also investigated through the
study of DNA degradation using reactors 1 and 2.
When a microorganism is not viable, this doesnt mean that its DNA has been dam-
aged. But if its DNA is strongly damaged, the microorganism is dead. This basic
consideration first motivated the study of the interaction of solutions of DNA (here,
both genomic DNA and plasmid) with the effluent of an argon discharge in order to
investigate the inactivation mechanisms induced by this plasma treatment.
A first set of experiments was conducted with reactor 2 for which gas flux was
not forced from the plasma zone to the contaminated surface; active species reached
the DNA contaminated surface through diffusion, possibly enhanced by the ionic
wind [15]. Distilled water droplets (10 mL) containing genomic DNA (1 mg) were
submitted to the effluent of a humid argon surface discharge during 10 min for three
discharge input power values: 2.5, 7.5 and 10 W. After collection, the treated DNA
solutions were analyzed by the gel electrophoresis technique (Fig. 8.6).
Lanes 3, 6 and 9 are controls. Lanes 1 and 2, obtained for the lower input power
(2.5 W), exhibit a smeared appearance, evidence of a partial degradation of DNA.
When the discharge input power is increased (7.5 W: lanes 4 and 5), the initial
DNA migration band disappears (complete degradation of DNA) and the smear is
shifted toward lower molecular weight. For the maximum input power (10 W:
lanes 7 and 8), the DNA is highly fragmented as shown by the weak smear in the
very low molecular weight migration region. In order to measure the hydrogen
peroxide concentration in the exposed droplets, the same experiments were per-
formed with distilled water instead of DNA solutions. As illustrated by Fig. 8.7
(reactor 2 data points and trend line), hydrogen peroxide concentration in water
droplets linearly increases with input energy (increasing input power for identical
102 E. Odic et al.
Fig. 8.6 Genomic DNA agarose gel electrophoresis after different treatment conditions. Lanes
19: 10 min. flowing post-discharge treatment (reactor 2: 2.510 W 0.5 L/min humid argon).
Lanes 1017: 10 min. incubation in H2O2 sol (S = samples, C = controls, migration from the top to
the bottom, .i.e. from high molecular weight to low molecular weight)
P (W) - Reactor 2
0 2 4 6 8 10 12
400
H2O2 in aqueous phase ppm
350
300
250
200
reactor 1
reactor 2
150
100
50
0
0 0,5 1 1,5 2 2,5 3
P (W) - Reactor 1
Fig. 8.7 Hydrogen peroxide concentration measured in 10 mL distilled water droplets after
10 min exposure time to the discharge effluent for increasing discharge input power. Reactor 1:
0.52.5 W 0.7 L/min humid argon. Reactor 2: 110 W 0.5 L/min humid argon
For the lowest value of input power, the DNA migration band first appears as
slightly smeared (0.5 W: lanes 1 and 2). Increasing the discharge power to 1 and
1.5 W leads to similar results (lanes 4, 5, 7, 8), but when a 2.5 W value is reached,
both the initial DNA migration band and smear have disappeared (lane 10), indicat-
ing an almost total fragmentation of genomic DNA.
Numerical simulation and experimental results previously reported [13] showed
that decreasing the gas flow rate in reactor 1 and thus increasing the residence time
in the discharge (which corresponds to an increase in the specific energy density and
also gas temperature), led to increased decomposition of the formed H2O2 for the
higher input power values. As a matter of fact, in Fig. 8.7 (reactor 1 data points and
trend line), hydrogen peroxide concentration vs. input power goes through a maxi-
mum value (~350 ppm) and then decreases. By comparing the results of Figs. 8.7
and 8.8, it can be seen that for the maximum H2O2 concentration, corresponding to
a 11.5 W input power range, the DNA degradation is low. However, when com-
plete DNA fragmentation is achieved (2.5 W), the H2O2 concentration is slightly
below 200 ppm, the same concentration which was found for a 0.5 W input power,
and for which DNA degradation was minimal. This indicates that a mechanism
other than reaction with hydrogen peroxide is needed to fully explain the observed
DNA damage, likely reaction with hydroxyl radical. Furthermore, an experiment of
10 min of incubation of DNA in H2O2 solutions did not show significant degradation
of DNA (Fig. 8.8 lanes 12 and 14).
Aside from oxidation by hydrogen peroxide, DNA degradation was then studied
with a lower molecular weight DNA: PET9SnI, a circular double stranded DNA. Its
three conformations can be seen in lane 1 of the gel electrophoresis photograph of
104 E. Odic et al.
Fig. 8.8 Genomic DNA agarose gel electrophoresis after different treatment conditions. Lanes
111: 10 min. flowing post-discharge treatment (reactor 1: 0.52.5 W 0.7 L/min humid argon).
Lanes 1215: 10 min. incubation in H2O2 sol (S = samples, C = controls, migration from the top to
the bottom, .i.e. from high molecular weight to low molecular weight)
Fig. 8.9: nicked open-circular (one strand cut) conformation, relaxed circular con-
formation and supercoiled conformation (covalently closed-circular 3D structure
resulting in a compact form). Plasmid solution (50 mg/mL) droplets (20 mL) were
exposed to the discharge effluent of reactor 1. For a constant input power (1.3 W),
the feed gas composition and flow rate were modified. Treatment times were
extended to 20 min. Submitted to a dry argon discharge effluent (lanes 36), the
migration band corresponding to the nicked open-circular conformation of plasmid
disappears, while the relaxed and supercoiled conformation migration bands are
strongly diminished in intensity; a smear is observed in the low molecular weight
region. It is worth noting that the effect is less pronounced for the lower flow rate
(0.2 L/min: lanes 5 and 6), probably caused by the increased transfer time for short-
lived species (mainly Ar* in this case). In the same conditions of flow rate, treat-
ment time and input power, dry argon was replaced by humid argon. A complete
fragmentation of plasmid was obtained, as can be seen in lanes 7 and 8 where neither
migration bands nor any smear can be observed. It can then be assumed that water
dissociation products are the main species responsible for DNA degradation, even if
excited argon may contribute (e.g. reacting with liquid water).
8 Investigations of Bacterial Inactivation and DNA Fragmentation 105
Fig. 8.9 Plasmid PET9SnI agarose gel electrophoresis after 20 min. flowing post-discharge treat-
ment (lanes 38). Reactor 1: 1.3 W 0.20.7 L/min dry and humid argon (M = molecular weight
marker, S = samples, C = controls, migration from the top to the bottom, .i.e. from high molecular
weight to low molecular weight)
8.4 Conclusion
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Naitali M (2009) Microbial inactivation using plasma-activated water obtained by gliding elec-
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charges for surface biological decontamination inside small diameter tubes. Plasma Processes
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The role of acidification for antimicrobial activity of atmospheric pressure plasma in liquids.
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the application of sterilization measurement and simulation of hydrogen, oxygen, and hydro-
gen peroxide formation. Int J Plasma Environ Sci Technol 1(1):96101
14. Dodet B, Odic E, Goldman A, Goldman M, Renard D (2005) Hydrogen peroxide formation by
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Chapter 9
DNA Oxidation by Reactive Oxygen Species
Produced by Atmospheric Pressure
Microplasmas
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 107
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_9, Springer Science+Business Media B.V. 2012
108 J.S. Sousa et al.
Reactive oxygen species (ROS) are well known to play an important role in several
biological systems, and generate oxidative damage to a variety of cellular components
[14]. If the amount of oxidative damage overcomes the repair capacity of the cell,
this can ultimately lead to cell death, which is very important to take into account
for biomedical applications of plasmas. Therefore, fundamental studies are essen-
tial to determine firstly the nature of plasma-generated ROS, and secondly the abil-
ity of those ROS to damage biomolecules. In this context, we have developed arrays
of microcathode sustained discharges (MCSDs) for the production of ROS at atmo-
spheric pressure. Recently, we have demonstrated that due to their remarkable sta-
bility, MCSDs can be operated in a continuous basis to produce DC glow discharges
in He/O2 mixtures, free from the glow-to-arc transition, at high gas pressure, with
low values of the reduced electric field (510 Td) and gas temperature (300400 K)
[5]. We have also shown that MCSDs are very effective in producing large amounts
of singlet delta oxygen (SDO) and ozone (O3) at atmospheric pressure [6]. In fact,
SDO densities higher than 1017 cm3 have been efficiently produced and transported
over distances longer than 50 cm, providing SDO fluxes greater than 100 mmol/h
[7]. Furthermore, O3 densities up to 1016 cm3 have also been obtained. More impor-
tantly, it has been shown that the density ratio of SDO to O3 can be finely and easily
tuned in the range 103 to 10+5 through the values of discharge current, gas flow and
mixtures (especially by adjusting the O2 and NO partial pressures) [6]. As so, these
arrays of MCSDs, by allowing a controlled production of either SDO or O3 at atmo-
spheric pressure, are ideal tools for studying in detail the reactivity of these ROS
towards biological components.
In the present paper, we used the molecule of deoxyribonucleic acid (DNA) as a
tool to monitor oxidative damage induced by plasma-generated ROS. Preliminary
results of experiments concerning the use of arrays of MCSDs as a plasma source
for biological studies concerning DNA oxidation are presented and discussed.
In order to study the reactivity of SDO and O3 towards DNA, aqueous solutions of
DNA were exposed to a gas flow of either SDO or O3. We have developed a
microplasma reactor using four MCSDs for the production of high SDO and O3
densities. As schematized in Fig. 9.1, each MCSD consists of a micro-hollow cathode
discharge (MHCD), a discharge concept first developed by Schoenbach et al. [8], and
a third 2.5 cm diameter planar electrode, positioned at 8 mm from the exit plane of
the MHCD. This 3-electrode configuration was initially proposed by Stark and
Schoenbach [9]. In our device, the MCSDs are powered from only one negative
power supply (for the four MHCDs cathodes) and from only one positive power
supply (for the four MCSDs anodes 2), through individual ballasting resistors. The
MHCDs anodes 1 are directly grounded. The distance between the individual
MHCDs is 8 mm. The MCSDs are operated in He/O2 mixtures (<4% O2) at atmo-
spheric pressure. It should be noted that to attain SDO densities higher than 1016 cm3,
low concentrations of NO (<200 ppm) have to be added into the gas mixture [57].
9 DNA Oxidation by Reactive Oxygen Species 109
As represented in Fig. 9.2, after passing through the plasma reactor, the gas flow
is evacuated through a line including a ROS-DNA interaction cell. The interaction
between the plasma-generated ROS (in gas phase) and the DNA (in aqueous solu-
tion) is performed at 55 cm downstream from the last MCSD. An accurate quantifi-
cation of the fluxes of ROS reaching the biological solutions is imperative for a
better understanding of the mechanisms of DNA oxidation. As so, we have mea-
sured and monitored the SDO and O3 densities at the entrance and exit ports of the
interaction cell [7], using infrared optical emission spectroscopy and ultraviolet
absorption spectroscopy, respectively, as described in detail in [6].
Oxidation of DNA has been performed by continuously bubbling aqueous solu-
tions of DNA with a gas flow of either SDO or O3. The biological experiments have
been conducted as a function of time of exposition, and under two very different
experimental conditions with respect to SDO and O3 densities, as shown in Table 9.1.
Indeed, in condition A, SDO density is maximal (1.5 1016 cm3) and O3 density is below
the limit of detection (<2.0 1012 cm3), while in condition Z, O3 density is maximal
(7.0 1015 cm3) and SDO density is below the limit of detection (<3.0 1013 cm3).
As DNA degradation is very sensitive to pH changes [10], buffered solutions
(10 mM KH2PO4 or 10/1 mM Tris-HCl/EDTA, pH = 6.8) were used in order to
maintain a constant pH. While the damages to the DNA backbone were analyzed by
agarose gel electrophoresis [11], the products of oxidation were detected and quan-
tified using the accurate and sensitive high performance liquid chromatography tan-
dem mass spectrometry method (HPLC-EIS-MS/MS) [12]. More details on the
experimental setup and procedure can be found in [7].
110 J.S. Sousa et al.
To evaluate DNA damage induced by SDO and/or O3, we have monitored the topol-
ogy of a plasmid DNA, pcDNA3.1 (Invitrogen), upon ROS exposure. Initially
pcDNA3.1 is mainly supercoiled (cf. Fig. 9.3, lanes 0), but ROS-induced single
strand break(s) (SSB) and double strand break(s) (DSB) lead to opened circular
(or relaxed) DNA and linear DNA, respectively. Plasmid DNA (Ci = 5 mg/ml) diluted
in 200 ml of either 10/1 mM Tris-HCl/EDTA (pH = 6.8) or of H2O (Cf = 20 ng/ml)
was exposed to the afterglow gas flow for different periods of time (up to 8 min). As
previously mentioned, damage to the DNA backbone of pcDNA3.1 was analyzed
by agarose gel electrophoresis. The results presented in the next sections are pre-
liminary and only a qualitative analysis is done. For the moment, not enough data
have been collected to correlate the amount of damages on the DNA backbone to
the number of molecules of SDO and O3 reaching the interaction cell.
At first, plasmid DNA was exposed to an afterglow gas flow of either O3 or SDO in
buffered aqueous solutions (10/1 mM Tris-HCl/EDTA, pH = 6.8). As shown in
Fig. 9.3, both ROS cause damage to the backbone of plasmid DNA, as revealed by
the change in the supercoiling of pcDNA3.1 (from supercoiled to circular and lin-
ear forms). Moreover, the number of breaks in the backbone rises as a function of
exposure time. However, as for equivalent times of treatment the number of dam-
ages generated by O3 is significantly greater than that induced by SDO, O3 seems
to be much more effective than SDO on oxidizing plasmid DNA. Indeed, after
4 min of O3 exposure, no more plasmid DNA in the supercoiled form is detected,
showing that at least one single strand break has been generated per molecule.
Furthermore, after 1 min of O3 exposure, double strand breaks are also formed as
revealed by the presence of linear DNA (Fig. 9.3, top panel). In marked contrast,
even after 8 min exposure of plasmid DNA to SDO, supercoiled DNA molecules
are still observed on agarose gel, and linear DNA is not detected (Fig. 9.3, bottom
panel). These results suggest that O3 is very efficient at generating single strand
breaks, when compared to SDO, and that greater time of exposure increases the
9 DNA Oxidation by Reactive Oxygen Species 111
Fig. 9.3 Digital photographs of agarose gels showing the effect of O3 (top panel) and SDO
(bottom panel) on DNA topology at different times of plasmid DNA exposition to a gas flow of
each reactive oxygen species
probability of two single strand breaks being formed opposite to each other. It
should be emphasized that for exposure times as long as 8 min only the gas flow of
O3 induces DSBs.
9.2.1.2 Influence of pH
Figures 9.4 and 9.5 highlight the importance of using buffered solutions in order to
study ROS-induced DNA oxidation. Indeed, similar experiments to those described
in Fig. 9.3 were conducted in H2O instead of Tris-HCl/EDTA. Two different sources
of water were used: a commercially available one (Analychrom, Fisher Scientific
Labosi, France), of chromatography grade (pH 6.8), and a home-made one
(pH 5.5), obtained by passing tap water through a 0.22 mm filter and further purify-
ing it using an ultra-pure water system (Purelab prima, ELGA). The initial pH of the
plasmid DNA solutions (before interaction with a gas flow of ROS; t = 0 min), as
well as their pH after 2 min of exposure, was measured with an indicator paper with
a precision of 0.25.
At first, we found that the amount of damages in the backbone tends to be con-
siderably higher in H2O (Figs. 9.4 and 9.5), when compared to the use of buffered
solutions of Tris-HCl/EDTA (Fig. 9.3), and that the time needed to reach the same
level of breaks is much shorter, for both gas flows of SDO and O3. This may be, at
least partly, explained by the fact that DNA concentration is kept constant in all
112 J.S. Sousa et al.
It should be noticed that this induced acidification was observed after only 2 min
of exposure, and regardless of the main ROS present in the afterglow gas flow.
Given our experimental conditions (closed and controlled atmospheric environ-
ment), reactive species from the gas phase have to be considered as the cause of
liquid acidification. Many factors could be involved in the observed decrease of pH.
Hydrogen peroxide (H2O2) generated in the liquid phase could have lead to the cre-
ation of acidic H3O+ ions by reactions with water molecules [13], consequentially
decreasing the pH. On the one hand, O3 decomposes in water creating hydroxyl
radicals (OH) [14], which, by recombination, can generate H2O2 [15]. On the other
hand, SDO by reacting with water molecules can also generate H2O2 [16, 17]. The
other possibility for decreasing pH in condition A might be the formation in the
liquid phase of nitrous acid (HNO2) and nitric acid (HNO3) via NO2 generated in
the gas phase from the NO that is added into the gas mixture [18, 19]. Moreover, we
cannot exclude that residual organic/inorganic compounds present in the H2O solu-
tions, even if at very low concentrations, may participate in the acidification of those
solutions when exposed to a gas flow of O3 or SDO. Nevertheless, the acidity of the
aqueous solution seems to also play a major role. Indeed, exposure of plasmid DNA
to O3 or SDO gas flows in H2O solutions with initial pH value of 5.5 (Figs. 9.4 and
9.5, bottom panels), instead of 6.8 (top panels), not only gives rise to circular single
stranded fragments but also to extensive broken plasmid DNA molecules (Figs. 9.4
and 9.5, t = 2 min). The fragmentation of the plasmid DNA is responsible for the
almost homogeneous continuum that is observed. These experiments show that the
characteristics of the liquid solutions used (notably their pH) affect the nature and
amount of DNA damages induced.
At last, it must be pointed out that few backbone damages do not mean few DNA
damages because of the possibility of direct oxidation of DNA bases (or even their
removal). As so, the analysis of the chemical modifications of DNA bases is essen-
tial for a better understanding of DNA oxidation.
In order to gain further insights into the mechanism of oxidation of isolated DNA by
SDO and O3, experiments have been performed concerning base chemical modifica-
tions. Buffered (10 mM KH2PO4, pH = 6.8) aqueous solutions (H2O and D2O) of
calf-thymus DNA (0.5 mg/mL in a total volume of 24 mL) were exposed to the
afterglow gas flow for various periods of time (up to 8 min). Calf-thymus DNA and
deuterated water (D2O) were obtained from Sigma (St. Louis, MO). Water was
deionized with a Millipore/Milli-Q system (Millipore, Molsheim, France). Prior to
the HPLC-EIS-MS/MS measurements, DNA digestions were performed as
previously described in [20, 21]. The main oxidation products of DNA bases
Adenine, Cytosine, Guanine, and Thymine are measured as 8-oxodAdo (8-oxo-7,
8-dihydro-2-deoxyadenosine), 5-OHdCyd (5-hydroxy-2-deoxycytidine),
8-oxodGuo (8-oxo-7,8-dihydro-2-deoxyguanosine), and DiolThy (cis- and
trans-5,6-dihydroxy-5,6-dihydrothymine), respectively.
114 J.S. Sousa et al.
As shown in Fig. 9.6, the main oxidation products of all DNA bases were
obtained when bubbling the aqueous solutions of DNA with a gas flow of O3.
DNA oxidation by O3 was, thus, successfully achieved. For the case of Adenine
and Thymine, the oxidized nucleosides production increases almost linearly
with the number of O3 molecules reaching the DNA solution. A two fold increase
in the number of O3 molecules leads to almost a two fold increase in 8-oxoAdo
and DiolThy. In the case of Guanine and Cytosine, we observe a biphasic curve
indicating that above a certain amount of O3 molecules, the formation of the
oxidized nucleosides starts to saturate. The stabilization of the number of
detected oxidized nucleosides is not likely to be related to a reduction of their
production rate, but instead indicates that these main oxidation products
(5-OhdCyd and 8-oxodGuo) might also be re-oxidized by the O3 flow. Therefore,
there might be a balance between their formation and destruction by O3.
According to the literature [22], the oxidized nucleosides 5-OHdCyd and
8-oxodGuo are rather susceptible to oxidation, which could explain the satura-
tion-like curve observed for those products. However, one should take into
account that a stabilization of the number of detected oxidized nucleosides
could also result from a deficient digestion of DNA. In fact, the enzymes respon-
sible for DNA digestion are less efficient when the number of oxidized bases is
relatively high, which is the case of these experiments where the number of
modified bases is considerable (0.13% of total bases). Nevertheless, the all
data suggest that O3 is very effective on oxidizing DNA, and especially Cytosine
(3% of Cytosine bases were oxidized).
Fig. 9.6 Amount of oxidized nucleosides (5-OHdCyd, DiolThy, 8-oxodAdo, 8-oxodGuo) per
million correspondent bases (Cytosine, Thymine, Adenine, Guanine) as a function of the number
of O3 molecules. Aqueous solutions of calf-thymus DNA were exposed to an afterglow gas flow
of O3 molecules for different periods of time up to 8 min. Samples were analysed by HPLC-
EIS-MS/MS
9 DNA Oxidation by Reactive Oxygen Species 115
According to Figs. 9.7 and 9.8, SDO seems to be also able to induce DNA oxida-
tion. In fact, all DNA bases but Cytosine are effectively oxidized by the SDO gas
flow. These results are somewhat in contradiction with the literature. The biochemi-
cal studies on the SDO-mediated oxidation of DNA that have been so far published
indicate that Guanine is the only normal DNA base that reacts with SDO [23, 24],
as no evidence of oxidation has been found regarding the other three bases. However,
Fig. 9.7 Amount of oxidized nucleosides per million correspondent bases as a function of the num-
ber of SDO molecules. Aqueous solutions (H2O) of calf-thymus DNA were exposed to an afterglow
gas flow of SDO molecules for different periods of time up to 8 min. Samples were analysed by
HPLC-EIS-MS/MS
Fig. 9.8 Amount of oxidized nucleosides per million correspondent bases as a function of the
number of SDO molecules. Same protocol as in Fig. 9.5, except for the use of heavy water (D2O)
instead of ordinary deionized water (H2O)
116 J.S. Sousa et al.
unlike what has been observed for a gas flow of O3, the amount of 8-oxodAdo and
8-oxodGuo generated by the SDO gas flow rapidly reach a plateau suggesting that
these oxidation products can also interact with the SDO gas flow and be further
modified. Additionally, the enhancing effect of D2O was used in order to confirm
the SDO-mediated DNA oxidation, as the SDO lifetime in D2O is assessed to be
525 times longer than in H2O [25, 26]. This H-D isotope effect on the SDO lifetime
is a consequence of an electronic-vibrational radiationless deactivation of SDO,
where the electronic excitation energy of SDO is converted into vibrational energy
of terminal bonds of deactivating collision partners. As the rate constant of this
energy-transfer mechanism is much higher with O-H bonds (2,900 M1 s1) than
with O-D bonds (132 M1 s1) [26], this leads to a considerably higher SDO lifetime
in D2O solutions. SDO molecules are, therefore, much less de-excited while passing
through a D2O solution than a H2O solution. For similar gas flows of SDO bubbling
the aqueous solutions of DNA, the SDO concentration is, thus, higher in D2O solu-
tions. As a consequence, the probability of a DNA molecule being oxidated by SDO
in D2O solutions is also higher. Indeed, in heavy water, about 530 times more dam-
ages were induced, and the plateaux were reached more rapidly, correlating the
oxidized nucleosides formation to the presence of SDO.
As shown in Table 9.2, in the experimental conditions that have been used in this
work, O3 molecules are one to three orders of magnitude more efficient than SDO
molecules on oxidizing DNA bases. In this regard, it is important to notice that, unlike
O3, most SDO molecules deactivate by physical quenching in the aqueous solutions
before reacting with DNA. In fact, the SDO lifetime in aqueous solutions is estimated
to be nine orders of magnitude lower than in the gas phase (~106 s) [26]. As O3 seems
to be much more effective on oxidizing DNA than SDO (cf. Table 9.2), O3 could still
play a role in oxidizing DNA in experimental condition A, even if its density is below
the limit of detection. However, taking into account that between experimental condi-
tions A and Z, the O3 densities differ from at least near four orders of magnitude, one
can conclude that, for all bases but Cytosine, at least ten times more damages were
obtained in experimental condition A than those which could be induced by residual
O3. This is a good sign for either direct or indirect SDO activity.
Table 9.2 Number of detected oxidized nucleosides per million correspondent bases for
experimental conditions A and Z, and the respective ratio
A Z
O3-induced damage
SDO ~ 1.5 1016 cm3 SDO < 3.0 1013 cm3 (Z) to SDO-induced
O3 < 2.0 1012 cm3 O3 ~ 7.0 1015 cm3 damage (A) ratio
8-oxodAdo 20 3,385 ~170
8-oxodGuo 30 833 ~28
DiolThy 40 4,689 ~117
5-OHdCyd 14 29,706 ~2,122
9 DNA Oxidation by Reactive Oxygen Species 117
9.3 Conclusions
The experiments that have been conducted indicate that SDO and O3 are able to
induce DNA oxidation, generating various damages in DNA such as single- and
double-strand breaks and oxidized bases. In particular, the number of modified
bases was considerably high (0.13% of total bases). It has been observed that while
all bases of DNA are almost indifferently and quite effectively oxidized by O3, SDO
reacts mainly with Guanine. Moreover, O3 seems to be much more effective on
oxidizing DNA. Indeed, double-strand breaks only occurred when using gas flows
of O3. Besides that, the amount of oxidized bases was also much higher when O3
molecules interacted with the DNA solutions, compared to the use of gas flows of
SDO. The enhancing effect of heavy water (D2O) has been used to confirm the
SDO-mediated DNA oxidation. When using heavy water, not only the same trends
have been observed as when using H2O, but also from 5 to 30 times more damages
were induced, correlating, therefore, the oxidized nucleosides formation to the pres-
ence of SDO.
The results that have been obtained, even if preliminary, are very significant and
demonstrate that arrays of MCSDs are a quite promising plasma source. We have
shown that arrays of MCSDs are very suitable and useful tools for biological studies,
and, thus, likely to lead to new biomedical applications. In fact, in the context of the
new field of Plasma Medicine, our plasma source is unique. Indeed, in contrast to
other available sources of ROS, our arrays of MCSDs are able to supply well-quantified
and tunable fluxes of either SDO or O3. Nevertheless, there are still many open ques-
tions on the reactivity of ROS with DNA. Our experiments have shown that the
characteristics of the liquid solutions of DNA play an important role, affecting
the amount and nature of the damages induced by the plasma-generated ROS on the
118 J.S. Sousa et al.
mechanical and chemical structure of DNA. In fact, the chemistry in the interface of
the gas and liquid phases is very important but rather complicated. For a better under-
standing of the mechanism of ROS-mediated oxidation of DNA, efforts are to be
made to gain further insights into the chemistry of the liquid phase.
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Chapter 10
Optical Emission Spectroscopic Evaluation
of Different Microwave Plasma
Discharges and Its Potential Application
for Sterilization Processes
Abstract The present work aims at studying different microwave flowing discharges
containing Ar and/or NO as alternative candidates to more extended N2 containing
plasma mixtures like N2-O2. Optical Emission Spectroscopy (OES) is used to dem-
onstrate the potential possibilities of these plasma mixtures to provide O* and UV
intermediate species demanded for sterilization purposes at low temperatures and
extended discharge gaps. Additionally, some plasma sterilization experiments with
Escherichia coli cultures are presented.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 121
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_10, Springer Science+Business Media B.V. 2012
122 J.L. Hueso et al.
10.1 Introduction
The dimensions of the quartz tube, terminated in a funnel-type enlargement, and the
stainless-steel cylinder chamber used for the experiments have been described else-
where [19]. It is coupled to the surface-wave (SW) surfatron which permits the
transformation of the electromagnetic power to the travelling wave and the propaga-
tion of the plasma discharge. The MW discharges are produced in a moderated
pressure range between 30 and 90 Torr, although the presented results are referred
to 45 Torr. Ar or N2 were fed through calibrated mass flow controllers as carrier
gases with a total flow of 50 cm3 min1. The concentration of NO and occasionally
O2 were kept constant at 3 103 and 2 104 ppm respectively. Axial profiles along
the x-axis of the quartz reactor at different points from the gap discharge have been
obtained for the main plasma mixtures. The OES spectra have been registered by
collecting the light with an optical fiber connected to a scanning monochromator
(Jobin-Ybon HR250) and a Hamamatsu photomultiplier (R928).
Escherichia coli DHa was grown in Lysogeny Broth (LB) liquid medium at
37C for 18 h. Prior to the plasma irradiation, the E. coli cells were extensively
washed with distilled water and the number of colony-forming units (CFU) was
determined by the viable count method to normalize the sterilization experiments.
This method was also used in the estimation of the survivor cells after the steriliza-
tion experiments. UV exposure experiments were carried out with a lamp emitting
at 254 nm. To properly compare the survival of E. coli after the different experi-
ments, total photon intensities measured by OES during the plasma experiments and
that of the UV lamp were used for normalization of results. Initial E. coli concentra-
tions were comprised between 1 108 and 3 108 CFU/mL (Fig. 10.1).
Fig. 10.1 Scheme of the experimental setup used for the OES study and the Escherichia coli
sterilization experiments: (a) mass flow controllers; (b) funnel-type quartz tube; (c) pressure
gauge; (d) MW power source; (e) surfatron launcher; (f) petri dish holder; (g) rotary pump/gas
outlet. (Right): digital photograph of the reactor
124 J.L. Hueso et al.
Figure 10.3 shows a typical OES spectrum of an Ar/NO plasma mixture and
Table 10.1 summarizes the main contributions and their electronic transitions. The
Ar atomic lines from the 5p excited state in the 700850 nm region and the Second
Positive system of N2* in the region 300400 nm are the most intense features
detected. Ar* lines from lower (i.e., 4p) excited states and some O* atomic lines are
also observed in this figure. The presence of O* and N2* can be explained by the NO
decomposition (Fig. 10.2).
A more detailed examination of the UV region in Fig. 10.3 shows the emis-
sion of NO* bands corresponding to the b and g systems. These species are espe-
cially relevant for sterilization purposes according to previous works with
low-thermal plasmas. The following reactions may resume some of the most
probable mechanisms, including a first excitation step and a subsequent de-
excitation process:
NO (X )+ e NO (A, B) NO (X )+ hn (a / b ) (10.2)
Table 10.1 Summary of main species detected by OES in the different plasma mixtures under
study [1921]
Species System Transition Position (nm)
N2 2nd Positive C3 B3 260400
NO b-System B2 X2 200300
g-System A2+ X2 260320
N2+ Principal 2
2 385395
Ar 4p and 5p lines 3s2p54p 3s2p54s 400440
3s2p55p 3s2p54s 700850
O Atomic lines 2s2p33p 2s2p33s 777; 782; 845
Fig. 10.2 Spectrum obtained by optical emission spectroscopy for an Ar/NO mixture (45 Torr,
60 W)
Fig. 10.4 (a) Influence of the distance from the resonant cavity on the emission intensities of NOg
and NOb intermediates in the Ar+NO plasma discharge. (b) Plot-Boltzmann of the Ar levels mea-
sured by OES from line intensities (45 Torr, 60 W) for the Ar-NO mixture. Red dotted line shows
the upper excited states employed for the calculation of the excitation temperature
explain the main dissociation pathways of NO and its relation with the electron
density [24]:
NO + e NO + + e + e (10.4)
Ar* + NO NO + + e + Ar (10.5)
NO + + e N + O (10.6)
Reactions (10.4, 10.5) are well known from the ionosphere chemistry and are
responsible for the production of the intermediate NO+ by electron impact ionization
(R5-6) or energy transfer from the Ar metastable states (R6). Depending on the posi-
tion of the glow discharge there is a competition between reactions (10.1, 10.2, 10.3)
for the production of NO and reactions (10.4, 10.5, 10.6) for its remediation. Recent
axial studies of MW Ar discharges at intermediate pressures have demonstrated the
rise of the electronic temperature at the end of the column [25, 26]. This fact can be
associated in our case with an increase of the recombination of molecular ions that
can contribute to the generation of NO* excited states through (R1-3) as we move
away from the surfatron launcher [2426]. From the point of view of the sterilization
efficiency, a higher concentration of NO* species at longer distances from the surfa-
tron launcher should ensure a higher sterilization efficiency even on surfaces not
directly exposed to the plasma. This difference supposes a clear advantage for the use
of Ar-NO for plasma sterilization.
The most common mixtures used in plasma based sterilization processes at low
pressures contain nitrogen as carrier gas. However, there are fewer studies in the
moderated pressure range considered in our case. The study of N2-NO and eventu-
ally N2-O2 will be mainly done for comparison purposes with the Ar-containing
mixtures. We find a first and clear distinction when we compare the OES general
spectra (Fig. 10.5) since the excited intermediates from the Second Positive System
of N2 are more intense than the others (Table 10.1). Conversely, there is no clear
signal from O* and only NO* intermediates from the NOg system are observed in the
presence of any of the other mixtures containing N2 as carrier gas (Fig. 10.6). This
fact is indicative of different excitation channels for Ar (atomic gas) and N2 (molec-
ular gas). In the case of N2 a great part of the energy transfer is consumed in the
excitation of vibrational levels. This fact might explain why only the less excited
levels of NO (i.e. NO(A)) are detected and why only the bands corresponding to the
NOg system are identified (Fig. 10.6a, b).
We underline reaction (10.7) as one of the main processes contributing to the
emission of NOg:
NO2* + NO(X) NO(A) NO(X) + hn(a ) (10.7)
128 J.L. Hueso et al.
Fig. 10.5 Spectrum obtained by OES for a N2/NO mixture (45 Torr, 60 W). The inset shows the
main transitions corresponding to the second positive system of N2
Fig. 10.6 Emission species in the UV region for: (a) N2-NO and (b) N2-O2 plasma discharges
In principle, the absence of the NOb system can be detrimental for sterilization
purposes in post-discharge configurations. Another major drawback accounts for
the limited length extension of the plasma discharge when N2 is present and the
abrupt decay of emission intensities as we move away from the surfatron launcher.
This difference supposes another clear advantage for the use of Ar-NO for plasma
sterilization. Not only the more active NOb species are formed with the mixture but
also a more effective exposure to UV emitters is ensured because of the higher
10 Optical Emission Spectroscopic Evaluation of Different Microwave 129
concentrations of NO* species existing at the end of the column. Nevertheless, the
presence of other chemical active species like long-live neutral O, especially in the
N2-O2 plasma mixture can not be ruled out for sterilization purposes [27, 28].
The plasma mixtures containing Ar seem to be really promising candidates for ster-
ilization purposes and their use is not so extended and studied [6, 14, 18] as the
low-thermal plasmas with N2 as main carrier gas. For this reason, we carried out a
sterilization experiment with Escherichia coli bacteria after exposure to Ar, Ar-NO
and N2-O2 plasma discharges for increasing periods of time. The survival curves are
depicted in Fig. 10.7.
Both the D-values (Decimal value) defined as the time necessary to reduce the
original concentration of micro-organisms by one order of magnitude [3] and the
sterilization kinetics present different trends for the different gases. After 300 s of
exposure to single feed Ar, the number of colony-forming units was 6-log10 reduced.
The curve present single-slope kinetics and D-value of 54 s. For the binary mix-
tures, there is a clear bi-phasic deactivation mechanism represented by a double-
slope survival curve with an initial D-value inactivation time (D1 < 10 s) much
shorter than the second one (50 s < D2 < 90 s). This behaviour has been previously
reported in N2-O2 experiments at low pressures but not for Ar-NO discharges. D1-
values are almost identical for both mixtures, although global results are slightly
better for Ar-NO. In principle, we attribute the observed differences to the activity
of UV radiation. This hypothesis is reflected in the survival curve found after expo-
sure of the E. coli cultures to the UV lamp (Fig. 10.7). The obtained curve defines a
similar trend to those of the N2-O2 and Ar-NO plasmas, thus supporting the impor-
tance of the UV radiation in the sterilization processes. The relatively lower effi-
ciency found for the UV experiment suggests that besides the effect of the UV
photons, other factors play a certain role in the case of N2-O2 or Ar-NO plasmas.
Indeed, SEM images in Fig. 10.8 show disruption and cell lysis of E. coli cells
subjected to Ar-NO plasma treatment. If we compare the untreated bacteria (c.f.
Fig. 10.8a) with the irradiated ones (Fig. 10.8b), we observe serious morphological
changes both lengthwise and transversely in many individuals. Therefore it is prob-
able that an additional etching mechanism from the Ar* and O* intermediates are
also contributing to the erosion of cells as proven by previous authors. From the
survival curves, it also arises that UV photons are the main contributing factors to
the damage of the bacteria since the UV lamp and the binary mixtures containing
NO* species exhibit a similar bi-phasic deactivation mechanism. The mono-phasic
trend observed for Ar* must be attributed to etching processes thereby corroborating
the active role of UV species and the suitability of Ar-NO plasma discharges to
provide a good combination of UV and etching species. Likewise, in the case of the
N2-O2 plasma mixture, an analogous synergetic effect combining UV radiation and
erosion from O neutral species is expected as previously addressed by Moisans
130 J.L. Hueso et al.
Fig. 10.7 Survival curve of Escherichia coli irradiated by Ar, Ar+No and N2+O2 surface-wave
plasma discharges and an UV lamp. Initial E. coli concentrations were comprised between
1 1083 108 CFU/mL. Adapted from [11]
Fig. 10.8 SEM images of Escherichia coli: (a) as untreated control and (b) after irradiation
with an Ar+NO plasma for 10 min. The inset in (b) is represented in an enlarged scale. Reproduced
with permission from [11]
research group [27, 28]. These afterglow atomic O species can not be detected in
our system but can be active in the VUV region and stable for microseconds, thereby
providing enough energy to break C-C bonds from DNA strands or cause oxidative
irreversible damage to proteins, cytoplasmic membrane and genetic material.
10.5 Conclusions
concentration of NO* (UV)-active emitters and Ar* and O* etching radicals that extend
up to much longer distances from the surfatron applicator than in the case of the
N2-NO counterparts. We put in evidence that the intensity of the NO* bands increases
at the end of the Ar-NO plasma while the NO destruction is maximum in the plasma
region close to the surfatron gap. We attribute this trend to the existence of two com-
peting reaction pathways where the electron density and the electronic temperature
are predominant at the beginning and the end of the plasma column, respectively.
Therefore, these factors should be taken into account for the design of sterilization
reactors in order to maximise the flux of active species. Likewise, it is shown that the
excitation of the vibrational and rotational levels of N2 limits its use as carrier gas and
constitutes a drawback for its implementation in sterilization processes of certain
dimensions or limited light of sight. In this sense, the use of a combined ternary
mixture of Ar-N2-O2 can be envisaged as a promising solution of compromise [14].
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NO/N-2/O-2, NO/CH4/N-2, and NO/CH4/O-2/N-2 systems by flowing microwave discharges.
J Phys Chem A 111(6):10571065
22. Yanguas-Gil A, Cotrino J, Alves LL (2005) An update of argon inelastic cross sections for
plasma discharges. J Phys D: Appl Phys 38(10):15881598
23. Yanguas-Gil A, Cotrino J, Gonzalez-Elipe AR (2004) Collisional radiative model of an argon
atmospheric capillary surface-wave discharge. Phys Plasmas 11(12):54975506
24. Dulaney JL, Biondi MA, Johnsen R (1987) Electron-temperature dependence of the recombi-
nation of electrons with No+ ions. Phys Rev A 36(3):13421350
25. Iordanova E (2010) Poly-diagnostic validation of spectroscopic methods: in-depth monitoring
of microwave induced plasmas. PhD Thesis, Eindhoven University of Technology, The
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26. Jimenez-Diaz M (2010) Modelling of microwave induced plasmas: the interplay between
electromagnetism, plasma chemistry and transport. PhD Thesis, Eindhoven University of
Technology, The Netherlands
27. Boudam MK, Moisan M, Saoudi B, Popovici C, Gherardi N, Massines F (2006) Bacterial
spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons
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N-2-O-2 mixtures. IEEE Trans Plasma Sci 30(4):14291436
Part II
Plasma Biofilm Inactivation
and Dentistry Applications
Chapter 11
Battling Bacterial Biofilms with
Gas Discharge Plasma
Abstract Most studies dealing with growth and physiology of bacteria have been
carried out using free-living cells. However, most bacteria live in communities
referred to as biofilms where cooperative interactions among their members make
conventional methods of controlling microbial growth often ineffective. The use
of gas discharge plasmas represents an alternative to traditional decontamination/
sterilization methods. We studied biofilms using two organisms, Chromobacterium
violaceum and Pseudomonas aeruginosa. With the first organism we demon-
strated almost complete loss of cell culturability after a 5-min plasma treatment.
However, additional determinations showed that non-culturable cells were still
alive after short exposure times. We have recently reported the effect of plasma on
P. aeruginosa biofilms grown on borosilicate coupons. In this paper, we present
results for plasma treatments of 1-, 3-, and 7-day old P. aeruginosa biofilms grown
on polycarbonate or stainless-steel coupons. Results indicate nearly 100% of
biofilm inactivation after 5 min of exposure with similar inactivation kinetics
for 1-, 3-, and 7-day-old biofilms, and for both materials used. The inactivation
A. Zelaya
Biological Sciences Department, California State Polytechnic University,
3801 W. Temple Ave., Pomona, CA 91768, USA
K. Vandervoort
Physics Department, California State Polytechnic University,
Pomona, CA, USA
G. Brelles-Mario (*)
Biological Sciences Department, California State Polytechnic University,
Pomona, CA, USA
Center for Research and Development of Industrial Fermentations,
(CINDEFI, CCT LA PLATA-CONICET), Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, La Plata, Argentina
e-mail: gbrelles@csupomona.edu
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 135
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_11, Springer Science+Business Media B.V. 2012
136 A. Zelaya et al.
kinetics is similar for both organisms, suggesting that the method is useful regardless
of the type of biofilm. AFM images show changes in biofilm structure for various
plasma exposure times.
11.1 Introduction
Fig. 11.1 Transmission electron micrographs of C. violaceum cells detached from a 3-day old
biofilm after a 20-min plasma treatment (b) detail of the area indicated in (a) by an arrow
Fig. 11.2 AFM images of C. violaceum bacterial biofilm treated with gas discharge plasmas for
0 min (a), 5 min (b) and 60 min (c). The images are 5 5 mm scan areas
Pseudomonas aeruginosa 1-, 3-, and 7-day old biofilms were produced in batch
culture using the CDC biofilm reactor (BioSurface Tech., MT). The biofilms were
grown on polycarbonate and stainless-steel 12.7 mm diameter coupons in TSB
(Tryptic Soy Broth) at 37C with agitation. After the selected growth time, the
coupons were aseptically removed from the reactor and unbound bacteria were
removed by rinsing the coupons twice with sterile saline. Coupons were briefly air-
dried prior to being subjected to gas discharge plasma for various exposure times
(5, 10, 15, 30, and 60 min) under sterile conditions. A control without plasma treat-
ment (0-min exposure time) was included. After treatment, the coupons were placed
in a wet chamber and incubated with 50 mL of sterile saline for 10 min. Biofilms
were then scraped off the coupons and suspended in 1 mL of sterile saline, serially
diluted, and suspensions plated in duplicates on an agarized solid TSB medium.
Plates were incubated at 37C and evaluated for colony-forming-units (CFU) by
counting the colonies. Data (CFUs/mL) were transformed to percentages assigning
the control 100% of survival. Short-exposure time experiments (0, 1, 2, 3, and
5 min) were carried out as described above for 3-day old biofilms.
The gas discharge plasma was produced using a commercially available inductively-
coupled Atomflo 300 reactor (Surfx Technologies, CA) that delivers an atmospheric
plasma jet [15]. The reactor consists of two perforated rectangular plates separated
by a gap 1.6-mm across. The upper aluminum electrode is connected to a 100-W RF
power supply (13.56 MHz), and the lower electrode is grounded. The size of the
plasma showerhead is 0.63 cm wide by 2.54 cm across. The average electron tem-
perature in the plasma is 1.5 eV and the average electron density in the plasma is
about 2 1011 cm3. The density of ground state N atoms is about 1 1015 cm3 at the
exit of the source and out to 5.0 mm. It starts to drop off thereafter. No significant
ion or electron density exists outside the source [11]. For the experiments, an atmo-
spheric-pressure plasma jet was generated using a He flow of 20.4 L/min, a second-
ary gas flow (N2) of 0.15 L/min, and an input power of 35 W. Both gases were
industrial grade. The plasma applicator was mounted such that the showerhead was
4 mm away from the biofilm.
To test the temperature reaching the coupon during plasma treatment, coupons of
either polycarbonate or stainless-steel were exposed to plasma previously equilibrated
140 A. Zelaya et al.
for 5 min. A thermometer was placed directly on the coupon surface and the
temperature was monitored and recorded once a minute for 10 min. The maximum
temperature reached by the coupon was 35C, confirming that temperature is not
responsible for biofilm inactivation since P. aeruginosa is a human pathogen that
prefers to live a t 37C.
A virulence test was developed by modifying a protocol designed for the model plant
Arabidopsis thaliana (Hong BY, 2010 personal communication). The mid-vein of
surface-sterilized leaves of Romaine lettuce was inoculated with a suspension of P.
aeruginosa cells obtained after treating biofilms with plasma as indicated in Sect. 11.2.1.
Unexposed P. aeruginosa was used as a positive control. A leaf injected with saline
only as well as a leaf that had not undergone any injections were also included as con-
trols. Leaves were then aseptically placed into UV sterilized- plastic bags. The bags
were sealed, laid flat at 37C and incubated for three additional days. Photos of leaves
were taken on the fourth day to obtain qualitative measurement of leaf damage.
11 Battling Bacterial Biofilms with Gas Discharge Plasma 141
The percentage of remaining culturable cells vs. plasma exposure time for stainless-steel
or polycarbonate-grown P. aeruginosa biofilms is shown in Fig. 11.3. The percent-
age of culturable cells at time 0 is 100% and corresponds to the control without
plasma treatment. The figures show that there is an obvious decrease in the percent-
age of culturable cells vs. time regardless of the biofilm age and the abiotic surface
used to grow the biofilm. A similar time-dependence behavior was reported for
P. aeruginosa biofilms grown on borosilicate coupons [15]. Seven-day old biofilms
behave in a similar way and do not seem to be more resilient than younger biofilms.
Similar results were also reported for C. violaceum biofilms grown for 4 or 7 days
on polystyrene microtiter plates [3, 6, 7, 9]. In the case of P. aeruginosa biofilms,
the decrease in the percentage of cells is even more dramatic since there are almost
no culturable cells after a 5-min treatment with plasma and most of the inactivation
occurs in less than 1 min of exposure to plasma (Fig. 11.3 insets b-2). However,
experiments with C. violaceum were carried out with a different plasma reactor and
although the spectrometry showed the same type of radicals that might be causing
cell inactivation (presence of NO g-bands around 250 nm and an OH band around
309 nm) [3], the actual amount of each radical may vary. The type of plasma used
for these experiments produces visible emission but a pretty insignificant amount of
UV since there is no detectable emission in the range of 200300 nm [11]. The
plasma conditions chosen for our experiments were those that maximized OH and
NO emissions and produced stable plasma [3]. The effect of temperature on biofilm
inactivation was ruled out as described in Sect. 11.2.3 and in reference [15].
Figure 11.4 displays typical AFM images for P. aeruginosa biofilms grown on
electropolished stainless-steel coupons and treated with plasma for different expo-
sure times, as indicated in Sect. 11.2.
For each 40 40 mm image, a cross section is included. For each image, a removal
of any overall background tilt was performed. This procedure involved subtracting
a plane determined from the average slope between the top and bottom edges and
right and left edges of the scan. There are no obvious qualitative differences that we
interpret from these images. From the cross sections of the images, the overall thick-
ness of the biofilm can be determined as the distance between the lowest features
(the flat surface of the stainless steel coupon) and the highest features (the peak of
the biofilm). Examining these differences yields biofilm thicknesses of ~700, 650,
and 500 nm for the 0, 5, and 30 min plasma-treated samples, respectively. A more
precise method for quantifying the surface topography of the biofilms was employed
by examining the average height of the surface features as relative to the lowest
point in the AFM scan (assigned the value of zero height). Using this method for the
ten regions sampled for each of the control, 5, and 30 min plasma-treated samples
yielded mean values of the average heights of 443, 415, and 401 nm, respectively.
Therefore, the trend of reduction of average height after a long period of plasma
treatment (30 min) was consistent with the reduction of biofilm thickness seen in the
images of Fig. 11.4 and the reduction in culturable cells in Fig. 11.3. However, there
a 1-day old biofilms
100
Percentage of culturable
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
Exposure time (minutes)
stainless-steel coupons polycarbonate coupons
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
Exposure time (minutes)
stainless-steel coupons polycarbonate coupons
90
80
70
60
cells
50
40
30
20
10
0
0 5 15 30 60
Exposure time (minutes)
stainless-steel coupons polycarbonate coupons
100
90
80
70
60
cells
50
40
30
20
10
0
0 1 2 3 5
Exposure time (min)
stainless-steel coupons polycarbonate coupons
Fig. 11.3 Percentage of culturable cells vs. plasma exposure time. Pseudomonas aeruginosa bio-
films were grown on either polycarbonate (orange) or stainless-steel (blue) for (a) 1 day; (b) 3 days
and (c) 7 days; and subjected to plasma for various exposure times and processed as indicated in
Sect. 11.2. Results are the average of at least four independent experiments. Platings for colony
counting were performed in duplicates. The bars, when present, represent the standard error of the
mean. In some cases, the standard error of the mean is not visible because it falls within the size of
the error bar. (b-2) Inset to part b. Short exposure times experiment
11 Battling Bacterial Biofilms with Gas Discharge Plasma 143
I II III
Fig. 11.4 AFM images of P. aeruginosa bacterial biofilm treated with gas discharge plasmas for
0 min (column I), 5 min (column II) and 30 min (column III). Cross sections of each 40 40 mm
scan are included, with the location of the cross section indicated by a horizontal line on the
image
0.8
Cantilever Displacement (m)
0.6
0.4
0.2
0
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.2 0.4 0.6 0.8
0.2
Adhesive
Step 0.4
0.6
0.8
Sample Displacement (microns)
Fig. 11.5 P. aeruginosa biofilm force-displacement curve for the 30 min plasma-treated sample
on electropolished stainless steel coupon. Data for tip-sample approach (dashed line) and tip-
sample retraction (solid line) are shown. The negative displacement of the cantilever that occurred
due to tip adhesion to the biofilm upon retraction is designated as the adhesive step
was a wide variability in the average heights measured for each of the samples and
the reported mean values differed by less than one standard deviation.
Figure 11.5 shows a typical force displacement curve obtained on the P. aerugi-
nosa biofilm grown on stainless-steel coupons for the 30 min sample. The curve dis-
plays the same general features that were exhibited in all of our measured
force-displacement curves. Upon approach (dashed line), the tip encounters the sam-
ple surface at the origin of the graph, and deflects upward with a slope that increases.
Upon retraction (solid line), the tip roughly retraces the approaching curve with some
hysteresis. Upon further retraction (in the third quadrant of the graph), the tip adheres
to the surface until it breaks free, and the points retrace the approaching data along the
144 A. Zelaya et al.
1.400
Displacement Curve Slope 1.200
1.000
0.800
0.600
0.400
0.200
0.000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Sample Region
Fig. 11.6 P. aeruginosa biofilm force-displacement curve slopes for 0 min (control, first ten bars)
and 30 min plasma-treated samples (last ten bars). The height of each bar on the graph corresponds
to the mean slope of five curves obtained for each region. Error bars represent plus and minus one
standard deviation about this mean
negative x axis of the graph. For the purposes of analysis, two sections of the curve
were considered. For approaching, the slope of the curve for positive sample displace-
ments up to 0.2 mm was determined. For significantly higher sample displacements,
the data are less reliable, since the optical detection of the cantilever deflection
becomes increasingly non-linear. Others have performed similar analyses on bacteria,
employing force-displacement curves over comparable scales [17, 18]. For retracting, the
height of the adhesive step, as indicated in Fig. 11.5, was measured.
The slope of the force-displacement curves can be used to determine an elastic
constant or stiffness of the biofilm. The derivation involves the analysis of the
relative compression due to the contact between two effective springs, the cantilever
and the biofilm. The biofilm stiffness, S, is related to the unitless slope, m (canti-
lever displacement/sample displacement), and the cantilever spring constant, k,
through the equation [17],
m
S=k
1 m
Therefore, when using the same cantilever for comparisons between different
plasma treatments, relative changes in the bacterial stiffness is a function of the
slope of the force-displacement curves only.
Figure 11.6 shows comparisons between the initial slope of the force-displacement
curve for the 0 min treatment (control) and 30 min plasma-treated samples on the
electropolished stainless-steel coupons. Curves at ten regions for each sample were
obtained. There is an overall increase in the slope of the displacement curves after
30 min of plasma treatment versus the control sample. The results differ by just
slightly more than one standard deviation. Specifically, the mean slope value of the
ten regions measured for the control sample is 0.627 with a standard deviation of
0.274. For the 30 min-treated sample, the mean slope value for the ten regions mea-
sured is 0.990 with a standard deviation of 0.040. Therefore, this increase in the
slope is consistent with an increase in the stiffness of the biofilm. These results
11 Battling Bacterial Biofilms with Gas Discharge Plasma 145
1.4
Adhesive Step (m) 1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 3 5 7 9 11 13 15 17 19 21
Sample Region
Fig. 11.7 P. aeruginosa biofilm force-displacement curve adhesive step data for 0 min (control,
first ten bars) and 30 min plasma-treated samples (last ten bars). The height of each bar on the
graph corresponds to the mean adhesive step height of five curves obtained for each region. Error
bars represent plus and minus one standard deviation about this mean
differ from our earlier results on biofilms grown on glass coupons, where we found
a slight decrease in biofilm stiffness with 30 min plasma treatment [15].
From the same force-displacement curves obtained for the sample regions shown
in Fig. 11.6, adhesive step data were extracted and are displayed in Fig. 11.7.
It is apparent from this graph that there is a wide variability in adhesive step
values over the various regions of each sample. The mean adhesive step height of
the ten regions measured for the control sample is 0.642 mm with a standard devia-
tion of 0.350 mm. For the 30 min-treated sample, the mean adhesive step height for
the ten regions measured is 0.328 mm with a standard deviation of 0.249 mm.
Therefore, there is a reduction in adhesion with 30 min of plasma treatment. This
reduction with plasma treatment indicates that the biofilm would exhibit less adhe-
sion to surfaces, detering its retention. These results are consistent with our earlier
findings for samples grown on glass coupons [15]. Pristine polycarbonate coupons
had surface height variations on the scale of a few microns, precluding their use in
the AFM studies, which could not delineate between the features of the biofilm and
the substrate background.
We previously reported that Chromobacterium violaceum biofilm-forming cells
undergo sequential morphological changes after plasma treatment. Bacterial cells
may undergo modifications ranging from minimal changes to putative loss of cell
walls. In another contribution, we verified the relative roughness of cells by exam-
ining image cross sections and analyzing the standard deviation of the surface
height. These surface features are consistent with cells undergoing damage [9, 14].
The present study goes beyond those reports suggesting that the architecture and the
stability of the biofilm as a whole may be impacted by plasma treatment.
Figure 11.8 shows the results of the virulence tests on lettuce leaves. P. aerugi-
nosa is also a plant pathogen and it produces tissue damage. It is clear that the mid-
vein of leaves in panels a and d show no damage whereas both the leaf treated
146 A. Zelaya et al.
Fig. 11.8 Lettuce leaves were injected with P. aeruginosa biofilm-forming cells treated with
plasma for 0, 5 or 30 min (panels b, c and d respectively) as indicated in Sect. 11.2.
A control injected with saline (panel a) was included as a negative control
11.4 Conclusions
Our results clearly show that bacterial biofilms can be inactivated by using gas dis-
charge plasma. The architecture and the stability, together with cell culturabilty, are
impacted by the plasma treatment. These results are evidence of the potential of
plasma as an alternative sterilization method against biofilms.
One of the issues of more concern regarding plasma-assisted cell inactiva-
tion is that, in most of the cases, the lethality of plasma is assessed solely based
on the number of colonies that can be counted after the treatment. However,
bacterial cells can respond to one or more environmental stresses by entering a
viable-but-non-culturable (VBNC) state [1921]. When cells are VBNC they
are unable to produce colonies on an agarized medium but they are still alive
and may retain pathogenicity. Therefore, not counting colonies after plasma
treatment cannot be considered as an obvious indication that the cells are dead.
We developed a virulence assay using lettuce plants for P. aeruginosa biofilms
and preliminary results suggest that cells retain viability after short exposures
to plasma. Therefore, and in agreement with previously reported results with
C. violaceum [3, 6, 7, 14], viability experiments should always be carried out
before drawing the conclusion that plasma is useful to kill cells based solely on
measurement of culturable cells.
Acknowledgments This work was supported by the U.S. National Institutes of Health under
Grant SCORE SC3 # 1SC3GM088070-01. Funding for the AFM was provided by the National
Science Foundation Nanotechnology Undergraduate Education Program, award # 0406533.
11 Battling Bacterial Biofilms with Gas Discharge Plasma 147
Anna J. Zelaya was supported by a fellowship from the U.S. National Institutes of Health RISE
Program 2R25GM061190-05A2. Sandra Sue Lwin was supported by the U.S. National Institutes
of Health under Grant SCORE SC3 # 1SC3GM088070-01. We thank Dr. Nina Abramzon for
allowing us to use her plasma reactor.
References
17. Zhao L, Schaefer D, Marten MR (2005) Assessment of elasticity and topography of Aspergillus
nidulans spores via atomic force microscopy. Appl Environ Microbiol 71:955960
18. Oh YJ, Lee NR, Jo W, Jung WK, Lim JS (2009) Effects of substrates on biofilm observed by
atomic force microscopy. Ultramicroscopy 109:874880
19. Oliver JD (1993) Formation of viable but nonculturable cells. In: Kjelleberg S (ed) Starvation
in bacteria. Plenum Press, New York, pp 239272
20. Colwell RR, Huq A (1994) Vibrios in the environment: viable but nonculturable Vibrio chol-
era. In: Kaye T (ed) Vibrio cholerae and cholera: molecular global perspectives. Proceedings
of the American Society for Microbiology, ASM Press, Washington DC.
21. Rozak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment.
Microbiol Rev 51:365379
Chapter 12
Inactivation of Microorganisms in Model
Biofilms by an Atmospheric Pressure Pulsed
Non-thermal Plasma
12.1 Introduction
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 149
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_12, Springer Science+Business Media B.V. 2012
150 Y. Akishev et al.
Gas discharge generators forming axisymmetric plasma jets are used for remote
treatment of 2D or 3D objects. These generators can be divided into two groups
which fundamentally differ from each other by gas dynamic characteristics of
plasma jets formed. The fist group includes generators applying RF, DBD and DC
discharges and forming steady-state cold plasma jet. NTP created inside these gen-
erators is blown out continuously by flow of plasma forming gas at moderate veloc-
ity about of 30 m/s. The second group comprises the PPSG generators based on
use of pulsed-periodical high-current (around 300 A) spark excited in small cylin-
drical volume (typical sizes are 5 mm in a diameter and 5 mm in the length). In this
case, active plasma is produced with little portions of short duration. Plasma jet is
not formed due to blowing through the gap by plasma forming gas but because of
strong expansion of the gas rapidly heated by spark (due to that the operation of
PPSG is accompanied with a loud noise). Continuous blowing through is required
to recover the gap for the next pulse. Plasma jet (or plasma cloud) flying out the
generator is very hot and has high outlet velocity close to sound velocity. High gas
12 Inactivation of Microorganisms in Model Biofilms 151
Fig. 12.1 Sketches and electrical schemes of axisymmetric PPSGs for remote treatment at atmo-
spheric pressure developed by different authors: (a) [19]; (b) [20]; (c) [18]; SG is a compliment
spark gap (SG) in series to the main gas gap; (d) image of PPSG by [18], in the latter case diameter
of an activated area on target is 1 cm
Fig. 12.2 Variation of instant Tg and average Tg with a distance from the outlet nozzle
Fig. 12.3 Current and light characteristics of spark and HPC formed by PPSG. (a) Time behavior
of spark current and intensity (in a.u.) of total light emitted from HPC flying past narrow slit
located at 10 mm away from the PPSG outlet. (b) Optical spectrum of the light collected from the
area at the PPSG outlet. UV spectrum of the spark emitted in the axial direction through the hole
is similar to that observed from HPC (see b) but its intensity at least 1,000 times higher. Plasma
forming gas is air; electrodes are fabricated of steel. The recording devices are: oscilloscopes
Tektronix TDS 520C and TDS 2012; photomultiplier tube FEU-100; spectrometer AvaSpec-
2048FT-6-RM with a resolution of 0.100.15 nm in all spectral region
excited by collisions with vibration excited N2. In such a case we can assume that popu-
lation of low energy electron states is determined by vibration temperature (Tv) of nitro-
gen in the ground state. Because of high gas temperature in HPC, V-T relaxation goes
fast and Tv is close to Tg. So we can determine instant Tg in the HPC from Boltzmann
plot for Fe atomic lines. The results obtained are presented in Fig. 12.2a. Full circles
show instant Tg determined from registration of the 2+ N2 system to excite this system
in HPC we used an auxiliary microdischarge located in different points of the cloud
trajectory. Average Tg determined by K-type thermocouple is shown in Fig. 12.2b. One
can conclude the instant Tg in HPC is higher than average Tg at the target.
Figure 12.3 presents experimental data on characteristics of spark and HPC pro-
duced by PPSG. They are typical for N2 and air as working gases. Figure 12.3a
demonstrates two features of the PPSG: (1) amplitude of spark current pulse is
about of 500 A with typical width about of 4 ms; (2) the moving HPC has a steep
leading edge and a gentle tailing edge, i.e. gas dynamic impact is applied to a target
12 Inactivation of Microorganisms in Model Biofilms 153
(the microorganisms) upon arrival the HPC; average velocity of the leading edge
over distance of 10 mm is about of 100 m/s. Figure 12.3b shows the most intense
emission of the PPSG corresponds to UV radiation in Fe atomic lines. One can
see that there is emission of O and N atoms, too.
biofilms were washed off (using a sterile brush of boarish bristle) into tubes containing
a saline solution. Rinses were tenfold diluted and plated on agar to determine micro-
bial titers. The dishes were incubated in a thermostat overnight at 37C. The grown
colonies were counted.
For the second series of the experiments on antimicrobial plasma treatment, we
used biofilms that were grown on the surface of mild steel (20 7 1 mm) and poly-
propylene (10 10 0.1 mm) coupons. The coupons were provided by the Institute
Biopribor (Puschino, RAS). The coupons were placed into tubes filled with
medium 8E and inoculated with E. coli or B. subtilis overnight suspension.
Incubation was performed at 37C for 314 days, with shaking (200 rpm). In these
experiments we used the same nutrient media and PPSG operating parameters as for
the first series. After NTP treatment the biofilms were washed off (with use of a
sterile brush of boarish bristle) into tubes with a saline solution. Rinses were tenfold
diluted and plated on agar dishes to determine microbial titers. The dishes were
incubated overnight at 37C in a thermostat so that the cultures formed colonies.
The grown colonies were counted.
Fig. 12.4 Number of E.coli cells grown on FMH agar (a) and 8E agar (b) survived in biofilms vs
plasma exposure time. Biofilm age is a variable parameter: (a) 1 2-h old culture, 2 overnight,
3 2 days old, 4 3 days old. (b) 1 2-h old culture, 2 2 days old, 3 5 days old, 4 6 days old. Statistical
scattering of our results does not exceed 30%
Fig. 12.5 Number of B. subtilis cells (grown on: (a) FMH agar, (b) 8 E agar) survived in biofilms
vs plasma exposure time. Biofilm age is a variable parameter: (a) and (b): 1 2-h old culture,
2 overnight, 3 2 days old, 4 7 days old. Statistical scattering of our results does not exceed 30%
(the results for biofilms grown on medium 27 C are practically the same as those for
medium 8E). In general, young E.coli biofilms were highly susceptible (or less
resistant) to plasma action. Nevertheless young biofilm grown over 2 h on the
enriched FMH-agar was incompletely inactivated by plasma after treatment over
5 min in spite of its rather low original titer.
Although absolute values of surviving bacteria in media FMH, 27C and 8E dif-
fer, there is a common tendency in their viability vs plasma exposure time: the total
number of microorganisms sharply decreases (by 25 orders of magnitude) in 1 min
of plasma exposure, and after that curves of microbial inactivation take a form close
to plateau. A similar tendency was observed for B.subtilis biofilms (Fig. 12.5).
B. subtilis biofilms were found to grow slower compared with E.coli biofilms.
For example, microbial titers of biofilms at the age of 1 and 2 days were similar,
whereas the titer of the biofilm grown on FMH medium for 7 days was higher by
156 Y. Akishev et al.
Fig. 12.6 Number of E. coli cells (a), and B. subtilis cells (b) survived in biofilm grown on metal
and polymeric coupons vs plasma exposure time. Biofilm age is a variable parameter: (a) 1 and 2
metal coupon, 3 and 6 days old biofilm, 3 and 4 plastic coupon, 3 and 6 days old biofilm. (b) 1 and
2 metal coupon, biofilm grown for 1 and 2 weeks, 3 and 4 plastic coupon, biofilm grown for 1 and
2 weeks. Statistical scattering of our results does not exceed 30%
almost 3 orders of magnitude (Fig. 12.5a). Besides, B. subtilis biofilms are more
plasma resistant. Indeed, although total number of microorganisms in B. subtilis
biofilms grown on FMH medium was markedly lower than that of E. coli biofilms
under identical conditions, we failed to fully inactivate old B. subtilis biofilms.
While in contrast to the E. coli biofilms, young B. subtilis biofilms grown on
medium 8E for 2 h or overnight were fully inactivated when exposed to plasma for
3 min (Fig. 12.5b).
Fig. 12.7 (a) Dynamics of free nucleotides concentration in the supernatant vs NTP-exposure of
E.coli cell suspension at isotonic conditions. (b) Survivability of NTP-treated E.coli liquid culture
incubated in the solutions with different osmotic pressure: 1 isotonic solution, 2 hypotonic solu-
tion. Statistical scattering of our results does not exceed 30%
12.5 Discussion
It was shown the 1-day (young or vegetative) biofilms are highly susceptible,
whereas the 3-day (old) biofilms are highly resistant to active plasma. The
decreased susceptibility of old biofilms is likely due to physiological ageing of
microorganisms, when the process of cell division is inhibited but both the amount
of accumulated cellular substances and strengthening of the cell wall increase.
Besides, the old biofilms may contain a significant amount of the dead cells
screening the living cells against plasma action.
It was revealed also that culture nutrient (enriched or poor medium) influences
drastically the viability of the cells injured by active plasma species. We associate
this fact with an existence of reparation processes inside the injured cell the cell
tries to repair the injuries delivered with active plasma species. The reparation goes
more effectively, if the injured cell is immersed in the enriched nutrient medium.
This process was discussed and taken into consideration in the mathematical model
of plasma-cell interaction published in [18].
The main feature of the pin-to-ring PPSG is that this plasma source activates an
object to be treated not continuously but periodically with intensive plasma clouds
(bullets). An important question needed to discuss is a spectrum of active agents
generated by PPSG developed. We distinguished three periodical stages in opera-
tion of the PPSG forming hot plasma bullets. These stages differ from each other in
types of active agents which can be responsible for remote inactivation of microor-
ganisms. In such an event, we can split each period in operation of the PPSG also
into three stages in bio-inactivation.
First stage has a short duration of about 5 ms and is associated with an appearance
of spark. At the first stage, only intensive spark UV radiation interacts with the
12 Inactivation of Microorganisms in Model Biofilms 159
transient interaction of hot gas with the microorganisms. In that case, practically
there is no difference what sort of gas (air or N2) will transfer a critical amount of
thermal energy to the cells.
12.6 Conclusion
The results obtained demonstrate that atmospheric pressure NTP produced by pin-
to-ring PPSG is an effective means to inactivate the microorganisms including bio-
films. Bio-inactivation by the PPSG is produced by pulsed periodical shooting the
targets to be treated by intensive UV flashes and hot and chemically active plasma
bullets of short duration. In such an event, the microorganisms are exposed not
only to strong UV radiation and chemically active species but to high transient gas
temperature as well. Such complex impact differs cardinally from that acting at the
inactivation by generators forming steady-state cold plasma jet. We found out
practically the same inactivation efficiency of the PPSG, if the working gases are
N2 or air.
It is shown that NTP treatment of microorganisms in liquid leads to partial or
complete degradation of the cell cytoplasmic membrane, the cell wall strength and
partial or complete breaking the integrity of intra-cell components. We assume that
main NTP active species in liquids are OH-radicals. In our opinion these species can
destroy not only the outside cell components these radicals can penetrate into the
cell due to their small sizes and interact with intra-cell components as well.
In total one can conclude that NTP PPSG antimicrobial treatment differs benefi-
cially from conventional methods to control biofilms: no chemically aggressive
reagents are required, and the plasma inactivation process takes short time compared
to long-lasting conventional procedures. Promising NTP method of bio-decontami-
nation based on pin-to-ring PPSG has much potential for practice and could compete
against traditional methods. There is one drawback of this method it is a loud
acoustic noise generated by pin-to-ring PPSG under its operation. However this
problem can be easily solved by using the proper sound isolation of the PPSG.
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sphere uniform glow discharge plasma (OAUGDP) for sterilization of surfaces and materials.
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11. Laroussi M (2002) Nonthermal decontamination of biological media by atmospheric-pressure
plasmas: review, analysis, and prospects. IEEE Trans Plasma Sci 30:14091415
12. Akishev Yu, Grushin M, Karalnik V, Petryakov A, Trushkin N (2010) Non-equilibrium con-
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17. Moisan M, Barbeau J, Moreau S, Pelletier J, Tabrizian M, Yahia LH (2001) Low-temperature
sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation
mechanisms. Int J Pharm 226:121
18. Akishev Yu, Grushin M, Karalnik V, Petryakov A, Trushkin N, Kholodenko V, Chugunov V,
Irkhina I, Kobzev E, Zhirkova N, Kireev G (2008) Atmospheric pressure non-thermal plasma
sterilization of microorganisms in liquids and on the surfaces. Pure Appl Chem 80:1953
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flow control actuator. AIAA meeting paper, Portland, USA, June 2004, pp 20042131
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Chapter 13
Low Temperature Atmospheric Argon Plasma:
Diagnostics and Medical Applications
Abstract This study was devoted to diagnostic of low temperature plasma produced
by microwave generator and investigation of its bactericidal effect against bacte-
ria in biofilms and within eukaryotic cells. The profile of gas temperature near
the torch outlet was measured. The spectrum in a wide range of wavelengths was
derived by the method of optical emission spec-troscopy. Probe measurements of
the floating potential of plasma were car-ried out. The estimation and adaptation
of parameters of plasma flow (tem-perature, velocity, ion number density) accord-
ing to medico-technical requirements were produced. The model of immersed
surface-associated biofilms formed by Gram-negative bacteria, Pseudomonas
aeruginosa and Burkholderia cenocepacia, and Gram-positive bacteria,
Staphylococcus aureus, was used to assess bactericidal effects of plasma treat-
ment. Reduction in the concentration of live bacteria in biofilms treated with
plasma for 5 min was demonstrated by measuring Live/Dead fluorescent labeling
and using direct plating. The intracellular infection model with the pathogenic
bacterium, Chlamydia trachomatis, was used to study the efficacy of microwave
argon plasma against intracellular parasites. A 2 min plasma treatment of mouse
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 163
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_13, Springer Science+Business Media B.V. 2012
164 S. Ermolaeva et al.
13.1 Introduction
Despite the large societal impact and economic burden, the problem of antimicro-
bial therapy of chronic infections remains largely unsolved [1]. Causative agents of
chronic infection are often resistant to the standard antimicrobial treatments. Besides
wide spreading of bacterial strains with multiple resistance to antibiotics, changes
in bacterial physiology in the course of prolonged infection affects sensitivity to
antimicrobial chemotherapy. One possible mechanism of the resistance of chronic
infections to antibiotics is the involvement of bacterial biofilms, a multilayer struc-
ture that includes bacteria and exopolysacharide matrix, and makes bacteria resis-
tant to conventional treatment strategies [2, 3]. Another evasive mechanism adopted
by some pathogenic microorganisms is through occupying intracellular niches,
which can be classified as intracellular parasitism [4, 5].
Physical methods of antibacterial treatment might be useful when traditional anti-
biotic therapy fails due to natural pathogen resistance or forming of specific niches
such as biofilms or intracellular vacuoles. Low temperature plasma is attractive for
such applications because (1) it possesses bactericidal activity; (2) the mechanism of
plasma antibacterial action seems to be independent of metabolic processes in
bacterial cells, and therefore it should be bactericidal for metabolically inert bacteria
in biofilms; (3) it has relatively low toxicity for eukaryotic cells and tissues [6, 7].
One of the purposes of this work was a development of methods for optical and
probe diagnostics of low temperature plasma. The plasma was produced by micro-
wave generator with frequency of 2.45 GHz at low power (~ 150 W) and with argon
as the feeding gas. Another aim was to evaluate the potential of low temperature
plasma as an antibacterial agent in therapy of chronic infections. Bactericidal activ-
ity of low temperature microwave argon was studied against bacteria in biofilms and
bacteria within eukaryotic cells, i.e. in conditions that modeled physiological status
of bacteria in the course of the chronic infection.
Biofilms are communities formed by surface attached bacteria [8, 9]. Biofilms
starts with bacterial adhesion, implemented by interaction of bacterial pili with a
surface. Attached bacteria multiply to form microcolonies. Further growth results in
a multilayer structure covered by exopolysacharide. Most biofilms exhibit some
level of heterogeneity in that patches of cell aggregates are interspersed throughout
an exopolysaccharide matrix that varies in density, creating open areas where water
channels are formed. Lower layers of the mature biofilm are not easily accessible to
chemical agents. Moreover, a part of the bacterial population is metabolically inert
that makes it resistant to antibiotics [8].
13 Low Temperature Atmospheric Argon Plasma 165
Fig. 13.1 Main stages of the C. trachomatis life-cycle. Infection starts when extracellular elemen-
tary bodies (EBs) enter the mammalian cell. Upon entry, the EBs turns into reticulate bodies (RBs)
and transform the phagosome into a replicative vacuole, called an inclusion. RBs multiply within
the inclusion until back transformation occurs into EBs. The infectious cycle is repeated upon host
cell destruction
life-cycle includes two forms, infectious extracellular elementary bodies (EBs) and
vegetative intracellular reticulate bodies (RBs) (Fig. 13.1). Intracellular RBs multi-
plying within intracellular vacuoles called inclusions are susceptible to antibiotics
that cross host cell membranes. In contrast, EBs are non-vegetating and metaboli-
cally inactive, being highly resistant to antibiotics and other medications.
Chlamydiae actively interacts with host signaling and metabolic pathways to
induce its own uptake by the host cell, obtain access to intracellular nutrients, and
maintain the integrity of the host cell until EBs become mature [6]. Chlamydiae
manipulate signaling pathways to hinder the activation of innate immune responses
detrimental to bacterial or host survival. In particularly, Chlamydiae have been shown
to inhibit host cell apoptosis and manipulate intracellular signaling systems driven by
transcriptional regulators, p53 and NF-kB [17]. Intracellular survival of Chlamydiae
is strictly dependent on manipulation by the host resources and any interruption of
the host signaling pathways can be vitally important for the pathogen. To study effec-
tiveness of low temperature argon plasma against intracellular bacteria, the model of
C. trachomatis infection of the murine fibroblasts McCoy was used.
All experiments were performed with the MicroPlaSter b device (Fig. 13.2) that
produces microwave plasma [18]. The MicroPlaSter b device allowed the research-
ers to use two regimes an argon plasma and a placebo regime with a flow of
13 Low Temperature Atmospheric Argon Plasma 167
non-ionized argon gas. The plasma source consists of a 2.45 GHz microwave
power supply, a torch, and a gas supporting system. The torch is a concentric tube
device, which is oriented vertically. The torch design causes a pattern required in
the plasma gas flowing through the torch tubes and entering the plasma. The micro-
wave power is coupled into the plasma torch via a coaxial cable. The torch should
generate a highly stable plasma jet operated at low powers (60150 W) with low
gas flow rates (48 l/min). Diameter of a plasma flow in such conditions is not less
than 30 mm. Inert gas consumption controls is carried out by system of monitored
inflow. Speed of argon flow defines a length of plasma torch which was about
3545 mm in our experiments. To measure such plasma parameters as temperature,
floating potential, densities of plasma components, we chose characteristic regime
of plasma torch for biological experiments with microorganisms and mammal tis-
sue (power of the discharge 120 W, argon flow velocity 5 l/min, orifice gas
argon with purity of 99.998%). The plasma torch was fastened on an optical table
with possibility of its exact positioning in space by means of micrometric screws
(Fig. 13.3).
The room temperature was about 19C. We measured gas temperature in plasma
behind torch outlet by the shielded thermocouple to exclude distortions of measurement
168 S. Ermolaeva et al.
Fig. 13.4 Gas temperature distribution in plasma behind the torch outlet
Fig. 13.5 Filters for control various components generated in a plasma torch
To obtain the quantitative data on plasma source composition the spectral diag-
nostic was used (Fig. 13.6). However, response of a spectrometer was too small for
definition of other active species in a plasma flow. It was offered to use for diagnos-
tics a special configuration of a torch with vertical windows for carrying out mea-
surements in the field of plasma generation and obtaining the intensity space
distribution in spectral lines of elements in vertical section of a plasma torch.
The results of spectrum measurements of a glow intensity distribution of an
active species in the plasma torch in vertical section are presented in Fig. 13.7.
As one can see in Fig. 13.8, the intensity and density of active species signifi-
cantly decreases out of generation area. However, the tests obtained by means of gas
analyzer MX2100 on distance of 20 mm for edge of the torch have shown concen-
tration O3 ~ 1,2 ppm, NO < 1 ppm, NO2 ~ 2,1 ppm that are substantial for sterilization
process of biological objects.
170 S. Ermolaeva et al.
Fig. 13.7 Results of spectral measurements of a glow intensity distribution for active species (Ar)
in the plasma torch in vertical section
Fig. 13.8 Results of spectral measurements of a glow intensity distribution for active species (OH
and N2) in the plasma torch in vertical section
We designed special probe to measure floating potential of plasma. With the help
of it we obtained profiles of plasma potential along the torch. The probe was kept by
molybdenum holder. When measuring we regulated a position of torch to probe
from 0 to 45 mm. The profile of floating potential in plasma along an axis z is
13 Low Temperature Atmospheric Argon Plasma 171
presented on Fig. 13.9. Plasma (electron and ion) density in this area is estimated in
a range of 105106 cm3 that is sufficient for an effective charging of small biological
objects. Further, measurements had shown that in interval of 535 mm behind torch
outlet the potential of an electrode decreased to 5 V.
Based on the results described previously, we chose the optimal regime of plasma
treatment. The samples were treated with the plasma source at a distance of
2 0.2 cm to ensure that the gas temperature was 36 2C throughout the
experiments.
The pathogenic bacteria, which are the most common causative agents of nosoco-
mial and wound infections, Pseudomonas aeruginosa (the strain PA103),
Staphylococcus aureus (the strain Sa78), and Burkholderia cenocepacia (the strain
Bc46) were used as model microorganisms. The different species were used to
check sensitivity to plasma in species differed in cell wall structure (Gram-negative
vs Gram-positive). The B. cenocepacia strain Bc46 was used to perform the quanti-
tative assessment as this strain high biofilm biomass due to a specific mutation [19].
The formation of surface-associated biofilms was performed on coverglass slices placed
into microtiter plates as previously described, but with slight modifications [20]. In
brief, an overnight culture was diluted 1000-fold in Luria-Bertani broth (LB broth,
Difco/BD, Franklin Lakes, NJ, USA) and cultured without agitation at 28C for
172 S. Ermolaeva et al.
Fig. 13.10 Effect of low temperature argon plasma on viability of bacteria in biofilms. (a, b)
newly formed P. aeruginosa biofilms, (e, f) newly formed S.aureus biofilms. (c, d) mature
P.aeruginosa biofilms, (g, h) mature S. aureus biofilms. A, C, E and G control bacterial biofilms,
B, D, F and H biofilms treated with plasma for 5 min. Differential live/Dead dyes labeled alive
bacteria green and dead bacteria red
174 S. Ermolaeva et al.
Fig. 13.11 Bacterial survival after biofilm treatment with plasma for 5 or 10 min. Bacterial con-
centrations are shown as denary logarithm of colony forming units (CFU) in 1 ml of suspension.
The mean values and SD are shown for each of three independent experiments. Decimal dilutions
of samples were plated in triplicates
The model of C. trachomatis infection of the murine fibroblasts McCoy was used to
study effectiveness of low temperature argon plasma against intracellular bacteria.
Cells were grown in Dulbeccos modified Eagles medium (DMEM, Invitrogen, US)
supplemented with 10% Fetal calf serum (FCS, HyClone, Australia), 2 mM Glutamine,
4.0 mg/ml Gentamycin and 5.0 mg/ml Amphotericin B in the 5% CO2 atmosphere. The
C. trachomatis Bu 434/L2 strain was routinely propagated in McCoy cells and elemen-
tary bodies (EBs) were purified by Renografin gradient centrifugation. Purified EBs
were resuspended in the sucrose-phosphate-glutamic acid buffer (SPG) and store fro-
zen at 70C. Titers were determined by infecting a cell monolayer with decimal dilu-
tions of the stock suspension. All experiments were performed with the multiplicity of
infection (MOI) 2. Infected McCoy cells were cultivated at 37C in the 5% CO2 atmo-
sphere up to 48 h post infection (hpi). Intracellular C. trachomatis were visualized with
luminescent microscopy as follows: infected McCoy cells were fixed with 50% metha-
nol, permeabilized with 1% Triton X-100 and stained with the monoclonal antibodies
specific for the C. trachomatis Major outer membrane protein (MOMP), which were
conjugated with the fluorescent dye fluorescein isothiocyanate (FITC,NearMedic Plus,
Moscow, Russia). Fluorescence of inclusion-containing cells were examined and
photographed under Nikon Eclipse 50i fluorescent microscope at x1350 magnification.
13 Low Temperature Atmospheric Argon Plasma 175
Fig. 13.12 C. trachomatis intracellular infection of McCoy cells at 48 hpi. Bacteria were visualized
by staining with FITC-labeled monoclonal antibodies (green). The cells were stained red.
(a) Control infection. (b) Infection was interrupted by plasma treatment of infected cells at 24 hpi
To perform a quantitative assay, serial lysate dilutions were used for infection of intact
McCoy cells. Infection was calculated at 48 hpi for all dilutions.
Treatment with low temperature argon plasma was performed as follows: just
before treatment, the culture medium was removed and about a ~1 mm layer of the
medium was left to preserve cells from desiccation during the treatment (Fig. 13.12a).
Cells were treated with plasma for 2 min and fresh DMEM medium was added
immediately afterwards. The treated cells were left for the next 24 h at 37C in the
5% CO2 in air atmosphere to complete the infection cycle. The cells were studied
microscopically as detailed above or cell viability was determined as follows. The
cells were stained with the 0.5% methylene blue dye for 2 h, lysed with 0.5% sodium
dodecyl sulfate (SDS, Sigma, USA) and the optical density was measured at OD540.
In parallel, the same experiments were carried out with non-ionized argon gas. As
control, non-treated cells were used. All experiments were repeated at least three
times in duplicate.
The control untreated cells contained big bacterium-containing inclusions that
occupy a considerable proportion of the cell volume (Fig. 13.12a). In contrast,
discontinued aggregates of bacterial cells without distinct borders were observed in
24 hpi plasma treated samples instead of regular inclusions (Fig. 13.12b).
To measure the concentration of live infectious bacteria in treated samples, the
cells were disrupted by ultrasound and the intact McCoy cells were infected with
the lysates (Table 13.1). Lysates obtained from untreated cells were used as a con-
trol. Secondary infection demonstrated reduction in the concentration of viable
infectious bacteria after treatment by a 5 logarithms; therefore, plasma treatment
had a considerable impact on viability of intracellular bacteria.
The important question arose about viability of the host cells upon plasma treat-
ment. Methylene blue staining demonstrated that the concentration of viable McCoy
cells only slightly reduced at 24 h post treatment with plasma. The overall reduction
in the concentration of viable McCoy cells was 20 2% (the mean from three times
made in duplicate). Thus, the plasma effect on viability of intracellular bacteria
considerably exceeded the toxic effect on the host cells.
176 S. Ermolaeva et al.
Table 13.1 Concentration of infecting particles after treatment at different stages of the
Chlamydia trachomatis life-cycle
Treated
Chlamydia Non-ionized
forms Control Plasma treated argon gas UV treated
RBs at 24 hpi 2,0 107 IFU ml1 1,1 101 IFU ml1 1,2 107 IFU ml1 3,5 105 IFU ml1
13.5 Conclusion
biofilms and intracellular infection were used to study the efficacy of low tempera-
ture argon plasma to eradicate bacteria in conditions, which are model conditions
typical of chronic infections. Five min plasma treatment reduced concentration of
viable bacteria in biofilms by a factor of a 100. Both Gram-negative and Gram-
positive bacteria in biofilms were sensitive to argon plasma. Mature biofilms did not
prevent bacterial killing. Argon plasma treatment of McCoy murine cells infected
with the obligate intracellular pathogen, C. trachomatis, interrupted intracellular
infection and caused noticeable mortality among bacteria. The loss of viability
among host eukaryotic cells was several logarithms less than mortality among bac-
teria. The results suggest that low temperature plasma can be effective in therapy of
chronic infections.
Acknowledgements We thank ADTEC Plasma Technology Co. Ltd., and in particular Mr.
Urayama for their part in the development of the MicroPlaSter device. We highly appreciate the
help of BioMedes Ltd. in manuscript preparation. The work was supported by Russian Ministry of
Education and Science (grants 02.740.11.0310 and 14.740.11.0118) and the Russian Foundation
for Basic Research (grant 10-02-01428).
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Chapter 14
A Sub-microsecond Pulsed Plasma Jet
for Endodontic Biofilm Disinfection
Abstract A pulsed, tapered cylindrical plasma jet, several centimeter long and
<2 mm in diameter, has been generated by a concentric tubular device for root canal
disinfection. This plasma dental probe is typically powered with ~100 ns, 1 kHz,
multi-kilovolt electric pulses and filled with 5 SLPM (standard liter per minute) He/
(1%)O2 flow. We report here an in vitro study of the antimicrobial effect of the room
temperature plasma jet against monolayer Enterococcus faecalis biofilms on bovine
dentins. Resultant colony-forming unit counts were associated with changes in bac-
terial cell morphology observed using scanning electron microscopy (SEM) follow-
ing the treatment and control. Treatment of dentin discs cultivated with E. faecalis
monolayer biofilms with the plasma (average power 1 W) for 5 min resulted in
92.4% kill (P < 0.0001). Severe disruption of the cell membranes was observed
for the plasma treatment group, while the morphology of the cells remained intact
for the negative control group. In addition, a pilot ex vivo test was conducted to
examine the bactericidal effect of the plasma against saliva-derived biofilms culti-
vated in human root canals. Conspicuous biofilm disruption and cleared dentinal
surfaces were observed in the canal after the plasma treatment for 5 min. We
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 179
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_14, Springer Science+Business Media B.V. 2012
180 C. Jiang et al.
Fig. 14.1 (a) Schematic of the plasma dental probe, and (b) an image of the cold plasma jet
impinging into a human root canal
pulse generator was used to drive the plasma device. The inductive adder-based
pulse generator is able to generate up to 10 kV, 100 ns pulses at a rate from single-
shot to 5 kHz. Current and voltage were measured with a current monitor (Pearson
2877) and a high-voltage probe (Tektronix 6015A) in conjunction with a digital
oscilloscope. The average power of the plasma dental probe was obtained with inte-
gration of the product of the voltage and current waveforms. During treatment, the
average power was measured to about 1 W. A flow meter (Omega, FL-3839ST)
delivers He/(1%)O2 gas mixture (Prepared by Airgas) to the plasma dental probe at
a flow rate of 5 SLPM (Standard Liter per Minute).
The plasma jet impinging into a root canal is shown in Fig. 14.1b. The plasma
plume can be touched with bare hand without causing pain or burning. In an indepen-
dent study, the surface temperature of an instrumented human tooth under plasma
exposure for 15 min was recorded by a thin-film platinum resistance temperature
detector (Omega, TFD), fixed on the outside wall of the tooth. The temperature
increased from room temperature to 28C in 10 min, then stayed almost constant
between 28C and 28.5C for longer plasma exposure time. This indicated a steady
state of heat exchange was reached for the plasma-tooth system after 10 min. In addi-
tion, the gas temperature of the plasma plume was measured to be about 300 K or
27C with optical emission spectroscopy by comparing the measured emission spec-
tra of the 2nd positive system of N2 with simulated spectra [19].
Optical emission spectroscopy and ultraviolet (UV) absorption spectroscopy
were conducted to identify reactive chemical species contributing to the plasma
sterilization process [7, 19]. Emission spectra in the 200800 nm wavelength range
were obtained for the He/(1%)O2 plasma plume [7]. Emission lines from excited
nitrogen molecules, excited atomic oxygen, excited helium, and nitrogen ions were
observed. Among them, atomic oxygen, the most reactive species, is able to inacti-
vate cells and cause cell lysis by oxidation [20]. OH emission was not observed. No
significant UV emission was observed between 200 and 300 nm for the atmospheric
pressure plasma. In addition, UV absorption spectroscopy measured ozone density
in the order of 1015 cm3 [19].
182 C. Jiang et al.
Freshly extracted, intact bovine molars were used for the in vitro tests. 6 mm
diameter, 1 mm thick dentin slices from the teeth roots were prepared, following the
protocol by Haapasalo and Orstavik [21]. The dentin discs were kept in tap water
during procedures to avoid dehydration. The smear layer was removed and the sur-
faces of the dentin discs were polished to ensure smooth dentin surfaces with Garnet
fine sandpaper disc (0.75 OD, Moore Company, Inc. Dearborn. MI). For steriliza-
tion, the dentin discs were treated with 6% NaOCl for 10 min followed by autoclav-
ing (121C, 20 min). After preparation of the specimens, the dentin discs were
immersed in sterile PBS solution until use for biofilm colonization.
An in vitro bacterial biofilm model was followed for the biofilm cultivation [22].
Prior to inoculation, ten bovine dentin slices were individually placed in lids of
500 ml Eppendorf tubes, which were then glued into six-well plates (Greiner bio-
one, Monroe, NC). All wells and the samples were disinfected with 6% NaOCl
solution and rinsed with sterile ddH2O. 7 ml of Luria Bertani (LB) broth (BD
Diagnostic System, Sparks, MD, USA) were added to each well and incubated
overnight at 37C to verify complete sterility. Then all wells were inoculated with
1 ml overnight culture of E. faecalis (ATCC 29212), obtained from the American
Type Culture Collection, and incubated for 6 days at 37C without shaking, with
daily change of 5 ml media.
Appropriate Institutional Review Board (IRB) approval was obtained for human
teeth and saliva collection. Four freshly extracted human teeth were subjected to
standard endodontic instrumentation. The crowns were cut off at the cementum-
enamel junction with a diamond disc (Hyperflex double-sided, No. 911. Brasseler
USA. Savannah, GA, USA). The root canals were then cleaned and shaped with
ProTaper rotary files (F2 and F3) (Dentsply, USA). During the root canal shaping,
the irrigation protocol was as follows: 1 ml of 6% NaOCl after each instrumentation
procedure. After instrumentation was completed, the teeth were immersed in 17%
EDTA for 5 min and then transferred to 70% ethanol (to prevent contamination)
until use for biofilm colonization.
A protocol adapted from the in vitro biofilm model of Guggenheim B et al. [21] was
followed. Prior to use, the teeth were thoroughly rinsed with sterile ddH2O and subject
to autoclaving at 121C for 30 min. Saliva was collected and incubated in Todd-Hewitt
broth (BD Diagnostic System, Sparks, MD, USA) without shaking at 37C for 24 h. The
sterilized teeth were placed in a six-well plate and filled with 4 ml Todd-Hewitt broth.
The six-well plate containing the teeth was inoculated with 1 ml of pre-cultured saliva
biofilm and incubated for 4 days at 37C without shaking with daily change of media.
14 A Sub-microsecond Pulsed Plasma Jet for Endodontic Biofilm Disinfection 183
For treatment, teeth were placed 5 mm below the plasma dental probe nozzle. Two
of the teeth were subject to the plasma exposure for 5 min. The other teeth, serving
as the control, were subject to the gas flow at the same flow rate for the same amount
of time, but with the plasma switched off.
One of the control tooth specimens was stained immediately with LIVE/DEAD
BacLight (6 mM for SYTO nine stain and 30 mM for propidium iodide) for 10 min
in the dark and washed with PBS thoroughly. The tooth was fixed with 4% formal-
dehyde for 1 h at room temperature in the dark and split crosswise with the dental
burr and examined in the confocal laser scanning microscope (CLSM 5 Pa inverted,
Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA) by immersing the sample in
184 C. Jiang et al.
14.2 Results
Fig. 14.3 SEM: (a) monolayer E. faecalis biofilms cultivated on bovine dentin discs without
treatment (the negative control); (b) the same monolayer biofilms at higher magnification
(8,000): E. faecalis cells appear morphologically intact (arrow 1); (c) after the plasma treat-
ment (He/(1%)O2 plasma, 5 SLPM, 5 min); (d) the same plasma treatment group at higher
magnification (10,000): membrane integrity severely compromised or damaged cells were
mostly observed (arrow 1). Morphologically complete but deflated or dying cells were also
observed (arrow 2)
186 C. Jiang et al.
SEM images of the root canals for each case were taken at different locations and
with different magnifications (ranging from 36 to 20,000). The typical SEM
images of the gas flow-treated root canal (the negative control) are shown in
Fig. 14.4a and b. For the control teeth, biofilms covered the entire root canal surface,
as shown in Fig. 14.4a. Saliva biofilms in different development stages were
observed on the dentinal surfaces. Biofilm thickly embedded in exopolymeric matrix
is shown in Fig. 14.4b. Microcolonies with predominant morphotypes of filamen-
tous rods and cocci were observed. CLSM (at several regions with different magni-
fications) of the LIVE/DEAD BacLight-staining of the root after the control
experiment shows that the biofilm inside the root canal consists of cells with pre-
dominantly intact membrane integrity and hence viable bacteria. A representative
CLSM image of the biofilms from the apical region of the canal is shown in
Fig. 14.4c. In one of the plasma treated root canals (Specimen #3), the plasma plume
reached a depth of 1 mm in the root canal revealing a distinct zone of biofilm clear-
ance as shown in Fig. 14.4d. In the same canal, biofilms occupied the regions where
the plasma failed to reach. This produced a visible contrast line and clearance zone
for plasma-treated and non-plasma treated surfaces, as clearly evident in Fig. 14.4e.
A predominantly clean surface revealing histomorphologically intact and open den-
tinal tubules was observed, as shown in Fig. 14.4f. The SEM images of the other
plasma-treated root canal (Specimen #4) show less effective biofilm disinfection
compared to Specimen #3. Bacterial-free patches on the dentinal surfaces were
observed but no complete biofilm removal. Bacteria with disrupted cell walls coex-
ist among morphologically intact cells, as shown in Fig. 14.4g and h.
14.3 Discussion
The quantitative microbiological and the visual morphological effects of the non-
thermal plasma jet on monolayer E. faecalis biofilms were assessed in the in vitro
study. We used E. faecalis as our first-step and representative study of the anti-
biofilm effects of the plasma because E. faecalis has been used as a biological
marker in a great deal of endodontic studies [17, 23, 24] and persists even in hostile
environments where nutrients are reduced and in the presence of antimicrobial
agents [25, 26]. In the experiment, viable monolayer E. faecalis biofilms formed on
dentinal surfaces. The microbiological analysis showed that the plasma approach
achieved 92.4% kill. The SEM images imply that better results could be achieved if
the plasma jet scans through the entire surface to ensure complete coverage by direct
plasma exposure. Nevertheless, the SEM results clearly show plasma-mediated
E. faecalis biofilm disruption.
The pilot ex vivo study demonstrated the plasma-based technique for bacterial
biofilm disinfection in root canal systems. The test system we have chosen repre-
sents an exemplar, in that copious salivary biofilms were grown in the root canals,
so that we could extrapolate to a smear layer in a real root canal. For one of the
Fig. 14.4 SEM and CLSM: (a) a salivary biofilm-formed root canal after treatment of He/(1%)O2
flow at 5 SLPM for 5 min (the negative control); (b) detail of the boxed area in the previous image
(magnification, 565): the matrix-embedded salivary biofilm covers almost the entire length of the
root canal; (c) CLSM image of biofilms in the same control root canal: viable bacterial biofilms
were observed for the control specimen; (d) after plasma treatment for 5 min (specimen #3). Note
that the right half (the entrance) of the canal has a distinct zone of clearance with a visible vertical
line left to which biofilms are still present where the plasma failed to reach; (e) detail of the boxed
area in the previous image; (f) a typical plasma treated root canal surface (specimen #3), revealing
open dentinal tubules; (g) a plasma partially treated root canal surface (specimen #4). Integrity
severely compromised morphotypes of fusiform bacteria (arrows) were observed among morpho-
logically intact cells; (h) a region of the plasma treated surface (specimen #4) where the
membrane-disrupted cocci (arrows) were observed
188 C. Jiang et al.
tested samples, this biofilm was removed by plasma treatment so that a predominantly
clean surface, revealing intact and open dentinal tubules in the root canal, was
observed in the plasma treated region. The test results for the other sample, how-
ever, showed disrupted bacterial biofilms and partially cleaned dentinal surfaces,
caused by plasma treatment, but no complete biofilm removal. As the two tooth
samples were randomly selected, the canal parameters may vary, which resulted in
different canal diameters and complexities that favors the plasma treatment or vice
versa. The incomplete biofilm removal may be due to failed or partial entrance of
the plasma jet into both root canals. One canal may have smaller upper (near the
corona region) diameter compared to the other, which resulted in higher level of
difficulty of the plasma entrance into the canal. We are in the process of developing
and optimizing the plasma sources for generation of plasma plumes into the entire
root canal. It has been recently reported that such plasma jets consist of fast propa-
gating ionization fronts with better repeatability and directivity compared to corona
discharges [2729]. The presence of a tooth with a narrow aperture under the plasma
nozzle would perturb the gas flow and affect the ionization channel of the plasma
bullets. Consequently, the diameter of the plasma plume may appear to affect the
entrance of the plasma plume into the canal whose diameter is typical 2 mm. A
1 mm-diameter plasma needle has been recently developed to enter into and com-
pletely fill submillimeter Tygon tubes [30] as well as root canals. The biofilm disin-
fection results by the improved plasma source will be reported in the near future.
No emission peaks were observed for the spectral range of 200315 nm, indi-
cating UV radiation-induced direct DNA damage on bacterial cells was negligible
for this plasma jet, which agreed with the observations by others [31, 32] on atmo-
spheric pressure plasmas. In addition, both the measured gas temperature of the
plasma plume and the temperature of the dentinal surface under plasma exposure
showed that heat did not play a significant role during the bactericidal process.
The previously reported optical spectroscopic results [7, 19] showed that the
plasma generated reactive chemical species including atomic oxygen and ozone.
These reactive plasma species may cause enhanced oxidation and membrane
disruption of microbial biofilms, and thereby may contribute importantly to the
antimicrobial effects.
14.4 Conclusion
In this study, a sub-microsecond pulsed atmospheric pressure plasma jet was gener-
ated for endodontic biofilm disinfection. The plasma-mediated antimicrobial effects
against E. faecalis and salivary biofilms were conspicuously observed. The antimi-
crobial effects of the non-thermal plasma have shown a novel, plasma-based tech-
nique that can be a potential alternative or a supplement to existing protocols for
root canal disinfection.
14 A Sub-microsecond Pulsed Plasma Jet for Endodontic Biofilm Disinfection 189
Acknowledgements The authors thank Dr. Shawn Anderson for the donation of the tooth
specimens for the experiments. This work is supported by the National Institute of Dental and
Craniofacial Research (NIDCR), one of the National Institutes of Health (NIH) in the U.S.
Department of Health and Human Services.
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Chapter 15
Medical Plasma in Dentistry: A Future Therapy
for Peri-implantitis
Abstract Biofilm formation plays a major role in the pathogenesis of many oral
diseases especially in peri-implantits. To evaluate the anti-biofilm effect of different
plasma devices and processes we used different dental biofilm models: Candida
albicans, Streptococcus mutans, Streptococcus sanguinis, aerobe multispecies
human saliva and anaerobe plaque biofilms. After 10 min treatment we reduced the
biofilms by 5 log10 steps using dielectric barrier discharge (DBD) plasma.
Chlorhexidine is the gold standard antiseptic which achieved in the same time only
a 1.5 log10 reduction. All plasma devices (DBD or plasma jets) damaged the mem-
brane of the microorganisms but only etching plasma sources can remove the bio-
film as shown in CLSM micrographs. It is possible to improve the plasma process
using antiseptics like octenidine. This combination significantly reduced CFU val-
ues after 1 min plasma treatment compared to the plasma control. Beside the anti-
biofilm effect an additional effect of plasma is the contact angle reduction of different
I. Koban (*)
Unit of Periodontology, Policlinics for Restorative Dentistry, Periodontology
and Endodontology, Ernst-Moritz-Anrdt University, Walther-Rathenau-Str. 49a,
17489 Greifswald, Germany
e-mail: ina.koban@uni-greifswald.de
A. Kramer
Institute for Hygiene and Environmental Medicine, Ernst-Moritz-Arndt University Greifswald,
Walther-Rathenau-Strae 49a, 17489 Greifswald, Germany
e-mail: kramer@uni-greifswald.de
K.-D. Weltmann
Leibniz Institute for Plasma Science and Technology e. V. (INP Greifswald), Felix-Hausdorff-Str.2,
Walther-Rathenau-Strae 49a, 17489 Greifswald, Germany
e-mail: weltmann@inp-greifswald.de
L. Jablonowski T. Kocher
Unit of Periodontology, Dental School, Ernst-Moritz-Arndt University Greifswald,
Rotgerber Str. 8, 17475 Greifswald, Germany
e-mail: lukasz.jablonowski@uni-greifswald.de; kocher@uni-greifswald.de
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 191
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_15, Springer Science+Business Media B.V. 2012
192 I. Koban et al.
titanium implant surfaces from 90 to super-hydrophilic (<5). This can improve the
implant healing process. Thus in the future, plasma could be an interesting treat-
ment option in dentistry, especially in treatment of peri-implantits.
15.1 Introduction
Many diseases in the oral cavity are caused by bacteria. The most common infectious
diseases in humans include marginal periodontitis. It is caused by bacterial plaque that
can result in inflammation and bone shrinkage which eventually leads to tooth loss
(periodontitis) (Fig. 15.1) [1]. Even on dental implants, similar inflammatory reactions
can be observed. At present peri-implantitis is a well known problem in the focus of
dentistry. The inflammation of the peri-implant tissue is caused by a biofilm on the
implant surface, leading to bone loss and jeopardize the longevity of an implant
(Fig. 15.2). Thus, within 10 years after implant insertion the prevalence of peri-implan-
titis is about 20% [2]. The biofilm removal is of great importance and a prerequisite for
the re-osseointegration. There does not exist a generally accepted and successful
method for biofilm removal of titanium surfaces [3]. To promote the primary osseointe-
gration, implants are very rough. Mechanical methods for biofilm removal, as they are
available on teeth, should not be used because they damage the rough and elaborate
surface topography of implants and they do not reach the deep cavities and recessions.
The disinfection with irrigating solutions does not remove the adherent biofilm. The
problem is magnified by the complex geometry of the screw implant.
So far, no general treatment recommendations exist for the removal of biofilms
on titanium surfaces in peri-implantitis [3]. Possible methods of biofilm removal
with rotating diamond burs, laser or air abrasion devices are associated with a
change in the surface structure and topography. Further, the implant surface is con-
taminated by the decontamination method. The resulting smear layer and the modi-
fied surface topography do not permit re-osseointegration [4]. Therefore, the
regeneration of peri-implant bone after an intra-operative implant processing within
the therapy is not yet possible to predict.
The dentist is currently lacking facilities
which remove biofilms in vivo without an adverse change of the implant surface
in combination with
to promote osseointegration of affected implants via concomitant surface
modification.
Opportunities: The implant manufacturers are aware that a chemically active and
hydrophilic surface modification can promote the early healing process by a cellular
interaction in the first phase of wound healing. This type of surface modification has
been used for some time in the industrial production of implants [5] and leads to an
improved and more rapid tissue integration [6, 7]. If it would be possible, to achieve
15 Medical Plasma in Dentistry: A Future Therapy for Peri-implantitis 193
Fig. 15.1 X-ray image of a patient with periodontitis and tooth loss due to periodontal disease
Early on, the antimicrobial effect of plasma on planktonic pathogens was detected
experimentally [8]. For this purpose, test organisms were streaked on agar plates
and treated with plasma. After a short treatment time inhibition zones could be
detected (Fig. 15.3).
194 I. Koban et al.
Fig. 15.4 Used plasma sources for laboratory tests: (a) hairline-pen, (b) kINPen in endodontic
model, (c) volume dielectric barrier discharge, (d) hollow electrode dielectric barrier discharge in
wells, (e) etching plasma jet on titanium plates
In the mouth, bacteria live in biofilms. The pathogens are covered in an extracel-
lular polymeric matrix (composed of polysaccharides, proteins, lipids and nucleic
acids) which protects them from environmental and physical influences as well as
antimicrobial substances. Hence, commercial antiseptics are insufficient in biofilm
treatment because they do not penetrate into the biofilm deeply enough. Biofilm
etching processes using plasma could solve these problems.
It was shown that a significant reduction of microorganisms, their metabolic
activity and inhibition of growth is possible with different plasma sources depend-
ing on the application time [911]. In order to test various plasmas in dentistry,
several sources are available (Fig. 15.4).
15 Medical Plasma in Dentistry: A Future Therapy for Peri-implantitis 195
Fig. 15.6 CLSM micrograph of a Streptococcus mutans biofilm before (left, 100) and after
(right, 100) plasma treatment (etching plasma jet 1 min, Ar+1%O2). Dark grey areas are live
cells, bright grey areas are membrane damaged cells. Important are the black areas in the right
micrograph after plasma treatment. Here the biofilm was removed
Etching plasma sources that can remove biofilms are needed. This jet (Fig. 15.4e) is
characterized by a grounded ring electrode and a centre rod electrode inside a quartz
capillary. The rod electrode is connected to the power source via a matching network.
The applied overall electric power of 65 W was held constant [16]. For biofilm removal
we used 5 slm argon and 1% oxygen.
Because of the antimicrobial effect plasma could be an interesting option for
endodontic application. The recent development of a new plasma device enables the
treatment of narrow cavities such as root canals (Fig. 15.4a) [17].
4
Reduction factor in log10 (CFU/ml) No
Ar gas
Ar plasma
*
3
0
control chlorhexidine octenidine control chlorhexidine octenidine
Streptococcus sanguinis biofilm saliva biofilm
Fig. 15.7 Reduction factors of Streptococcus sanguinis and saliva multispecies biofilm after treat-
ment with chlorhexidine and octenidine in combination with a following argon gas and plasma
treatment (kINPen 09, 1 min) * P < 0.05 versus Ar plasma control
the antiseptics chlorhexidine and octenidine. Here we used the model organism
Streptococcus sanguinis, which is one of the first colonizers adhering to saliva-coated
human tooth surfaces or dental materials [19]. In this monospecies biofilm model,
the highest log CFU reduction factor was found for argon (Ar) plasma without any
admixture (negative control). As a more realistic model concerning peri-mucositis
we used a multispecies saliva biofilm. Our results for this multispecies biofilm
showed that we found synergistic effects between plasma and agents. The combina-
tion octenidine and plasma significantly reduced CFU values compared to the plasma
control. Here, we achieved a similar reduction factor as with the monospecies biofilm
using plasma without admixture (Fig. 15.7). Therefore, it is effective to combine
plasma with special antiseptics. Further investigations are necessary to clarify the
mechanism of this synergism. Additionally other antiseptics should be tested.
Ar Plasma
Ar + 0.2% O2 Plasma
Ar + 1% O2 Plasma
Contact angle in
Contact angle in
Treatment time in s Treatment time in s
Fig. 15.8 Effect of plasma treatment (kINPen09) on the contact angle of titanium
15.3 Summary
Acknowledgements This work was realized within the framework of the multi-disciplinary
research cooperation Campus PlasmaMed, particularly within the project PlasmaDent. The
authors acknowledge that this work was supported by a grant funded by the German Ministry of
Education and Research (BMBF, grant no, 13N9779). All titanium discs were kindly provided by
Straumann (Institut Straumann AG, Basel, Switzerland).
We thank Tina Dornquast, Claudia Lehnert and Hartmut Fischer for their excellent technical
assistance, Rdiger Titze for his skilful support in operating the plasma equipment and Karsten
Schrder for critical discussions as well as Christoph Schmidt and Sander Bekeschus for critical
reading of the manuscript.
15 Medical Plasma in Dentistry: A Future Therapy for Peri-implantitis 199
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202 W. Zhu et al.
Flow
Gas
100
Flow - HV
Gas
5k 9.0 cm
2.0 cm
1.0 cm 2.0 cm
Petri Dish
Fig. 16.1 Schematic diagrams of a DC atmospheric pressure non-thermal plasma microjet work-
ing in air and in water, and their corresponding pictures
power supply through a 5 kW ballast resistor and the outer electrode is grounded
for safety considerations. The nozzle opening of the plasma device is 0.8 mm in
diameter and approximately 1 mm deep. Details about this device and electrical
circuit can be found in references [21, 22]. Premixed He/O2 (2% in volume) was
used as the working gas at a flow rate of 2.5 slm (standard liters per minute).
Figure 16.1 shows a schematic diagram of the plasma device in air, in water and
their respective pictures. When sustained in air, the visible jet is ~25 mm at a
sustaining voltage about 400 V and a discharge current of 35 mA. When immersed
in water, the PMJ was sustained in a quasi-steady gas cavity with approximately
the same sustaining voltage.
Each Candida strain was grown for 2 days on sabouraud dextrose agar (SDA) at
35C to ensure the viability and purity.
When treated in air, 100 ml diluted suspension (1 104 CFU/ml) were spread
evenly onto sabouraud dextrose agar (SDA) via a sterile plastic loop. This initial
concentration was chosen on purpose so that all results from various treatment times
can be fit into the CFU count detection limit in a later stage. The plasma treatment
(visible portion of the plasma touches agar) was limited to a 2 2 cm2 square area
(referred to as treated area) at the center of a 9 cm diameter Petri dish (as shown
in Fig. 16.1), with a treatment time ranging from 0 to 10 min. CFU counts from
controls with only gas flow are within error range of that from samples without any
treatment. The exit nozzle of the PMJ is maintained at about 1 cm away from the
treated media, where temperature was evaluated to be below 40C. This is not a
temperature that can affect the survival of the fungi.
For the treatment in water, conidial suspension was diluted to a concentration of
(13) 106 CFU/ml. Twenty milliliter suspension was treated with He/O2 PMJ from 0
to 4 min. After the plasma treatment, 200 ml suspensions was aspirated out and further
diluted 1,000 and 100-fold to perform antifungal susceptibility test and colony count-
ing, respectively. All experiments were repeated 3 times for statistical analysis.
The percentage of inactivation (PI, defined as 100% (1 CFU treated /CFU control ) )
of the Candida strains treated in air and in water are plotted in Fig. 16.2. In air, in
the treated area on Petri dish (Fig. 16.2a), there is a fast increase of the PI for all four
Candida species within the first minute, followed by a much slower change of PI. It
appears that different strains respond to plasma treatment slightly differently, with
C. glabrata reaching 100% inactivation in 2 min, while C. krusei only reaching 91%
after a 10-min plasma treatment. We also evaluated the CFUs in the untreated area
on Petri dish. Interestingly enough, the Candida strains in the untreated area were
also inactivated. Although the initial change of the PI with respect to time in the
untreated area was not as fast as it was in the treated area, significant PI (~90%)
was observed after a 10 min plasma treatment (Fig. 16.2b). It is possible that long
lived reactive species (such as ozone) can laterally transport to the untreated area,
where critical dosage is reached with the increase of treatment time.
While these Candida strains were treated in water, a fast increase of PI to above
90% was observed within the first 30 s. A 100% inactivation was achieved around
1 min treatment, as shown in Fig. 16.2c. This fast inactivation in water is related to
free radical production and will be discussed in Sect. 16.5.
XTT colorimetric-assay (Sigma, St. Louis, MO) was used to evaluate the cellular
viability of Candida strains through measuring the activity of enzymes that reduce
XTT to Formazan dyes. After PMJ treatment, 20 ml of the sample was added into
100 ml XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car-
boxanilide) solution in a well of a 96-well microtiter plate. Each sample runs in
duplicate. The plates were then covered by aluminum foil and incubated at 37C for
2 h. The resulting supernatant was transferred via a multichannel pipette into the
wells of new microtiter plates, which were then read in a microtiter plate reader
(Bio-rad680). The optical density (OD) of the supernatant in each well was deter-
mined by measuring the absorbance at a wavelength of 490 nm. The OD of the
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 205
a 120 b
100
Percentage of Inactivation
Percentage of Inactivation
100
80
80
60
60 In Air
40
In Air (Treated Area) (Untreated Area)
40
C. glabrata 00271 20 C. glabrata 00271
20 C. krusei 00279 C. krusei 00279
C. albicans 002971 0 C. albicans 002971
0 C. albicans SC5314 C. albicans SC5314
20
0 2 4 6 8 10 0 2 4 6 8 10
Time (min) Time (min)
c 120 d 120
C. glabrata 00271
Percentage of Inactivation
0 1 2 3 4 0 1 2 3 4
Time (min) Time(min)
Fig. 16.2 Percentage of inactivation of C. glabrata (BMU 00271), C. krusei (BMU 00279),
C. albicans (BMU 02971) and C. albicans (SC5314) treated with He/O2 PMJ (a) in air in the
treated area, (b) in air in the untreated area, (c) in water and (d) XTT assay
treated samples was then compared to that of the control to generate a percentage.
The XTT results of planktonic Candida strains are plotted in Fig. 16.2d. It shows
fairly good agreement with the inactivation in water. Zero metabolic activity was
achieved within 1 min except for C. albicans (SC5314) where 4 min is needed.
Nevertheless, this indicates that these Candica strains were essentially not viable
after the plasma treatment.
Ten strains of Candida species isolated from different sources were used in the bio-
film study, as listed in Table 16.1.
After each strain was grown on potato dextrose agar (PDA) at 35C for 3 days to
ensure the viability and purity, a loopful of Candida cells was inoculated in 20 ml
yeast peptone dextrose liquid medium and incubated overnight in a shaker (150 rpm)
at 30C. Cells were then harvested from the liquid cultures by centrifugation
(3,000 g 5 min at 4C), and washed by ice-cold sterile PBS. RPMI-1640 medium
206 W. Zhu et al.
was used to re-suspend the pellet and adjust the final density to 1.0 106 cells/ml for
all strains. Hundred microliters of prepared suspension was pipetted into selected
wells of 96-well microtiter plates and every replication was separated by an empty
well. Biofilm was formed after incubation at 37C for 24 h. The medium was aspi-
rated in the wells which were then washed with sterile water three times to remove
planktonic cells. The biofilms were treated by PMJ for 10 s, 20 s, 30 s and 1 min,
respectively. A photo of the PMJ above the microtiter plate is shown in Fig. 16.3.
After PMJ treatment, 100 ml of sterile water was added into the well and washed
vigorously in order to re-suspend the biofilm cells. The suspension was then diluted
1,000 times with sterile water from which 100 ml was pipetted out and spread
evenly by a sterile plastic transferring loop onto SDA. The CFU counting was per-
formed after 24 h incubation at 35C. One hundred microliters RPMI1640 medium
followed by 100 ml of 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenyl-
Amino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT, 1 mg/ml, Sigma-Aldrich Co.,
St. Louis, USA) and menadione (10 mM, Sigma-Aldrich Co., St. Louis, USA)
mixture were added into the wells treated by PMJ. The plates were then covered by
aluminum foil and incubated at 37C for 2 h. Eighty microliters of the resulting
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 207
Percentage Inactivation
Percentage of Inactivation
80 80 80 80
XTT (%)
XTT (%)
60 C. krusei
60 60
C. glabrata 60
0 0 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (s) Time (s)
c 100
100
Percentage of Inactivation
80 80
XTT (%)
60 C. albicans 60
BMU 04801
40 BMU 02971 40
SC5314
20 BMU 03213 20
0 0
0 10 20 30 40 50 60
Time (s)
Fig. 16.4 Percentage of inactivation and cell viability test (XTT assay) of C. glabrata, C. krusei
and C. albicans biofilms (iosolates from different sources, see Table 16.1) by He/O2 PMJ
supernatant was transferred via a multichannel pipette into the wells of new micro-
titer plates, which were then read in a microtiter plate reader (Bio-rad680). The
optical density (OD) of the supernatant in each well was determined by measuring
the absorbance at a wavelength of 490 nm. Each group contained negative and
positive controls.
Figure 16.4 shows the inactivation and XTT results of C. krusei, C. glabraca
and C. albicans biofilms versus treatment time. Three important observations: (1)
different Candida biofilms (even the same strain but isolated from different sources)
respond to plasma treatment slightly differently; (2) all Candida biofilms are inac-
tivated by PMJ rather quickly, all reaching approximately 100% within 30 s. (3)
XTT assay corresponds well with CFU counts, indicating the loss of viability of
the cells.
C. albicans (SC5314) biofilms (with and without PMJ treatment) were fixed
overnight with 2.5% glutaraldehyde and then dehydrated in ethanol. The samples
were gold-paladium coated and evaluated with SEM (Quanta 200FEG and NOVA
NANOSEM 430). Figure 16.5 shows five SEM images of C. albicans (SC5314)
biofilms treated with He/O2 gas flow and with He/O2 PMJ for different time periods.
Magnifications of these images are at or around 10,000 times. Figure 16.5a shows
that healthy yeast cell and pseudohyphae are with smooth surfaces. With the increase
of the plasma treatment time, cell rupture, distortion and shrinking were observed
208 W. Zhu et al.
Fig. 16.5 SEM images of Candida albicans (SC5314) treated with (a) He/O2 gas flow, He/O2 PMJ
for (b) 10 s, (c) 20 s, (d) 30 s and (e) 60 s
(Fig. 16.5be). The biofilm lost its original morphological characteristics and
degraded to clusters of cell fragments.
Antifungal susceptibility test was performed in samples treated with PMJ following
The Clinical and Laboratory Standards Institute recommended reference standard
M27-A3 with minor modification [23]. Two major concerns that motivated this test are:
(1) some fungi may survive a certain dosage of plasma treatment. They are possibly,
however, modified by the plasma and are more susceptible to traditional antifungal
treatment. (2) Clinical trials often combine traditional treatment methods and the
developing technique. As the toxicity and safety study of the plasma treatment is still
lacking, one might want to reduce the exposure to plasma to a minimum dosage while
achieving a considerable reduction of the dosage of traditional antifungal therapy.
Three commonly used antifungal drugs, namely fluconazole (Fuyang Genebest
Chemical Industry Co. Ltd, China), caspofungin (Merck, NJ, USA), and amphotericin
B (Sigma-Aldrich Co., St. Louis, USA) were used in the antifungal susceptibility
tests. From the stock solution of each antifungal agent, final working concentrations
were prepared in RPMI1640 medium to be 2561, 20.015 and 40.125 mg/ml,
respectively. Hundred microliters of diluted the respective solutions were added into
wells containing biofilms. After incubation at 37C for 24 h, their metabolic activi-
ties were evaluated with XTT assay as described above. Candida parapsilosis ATCC
22019 was included as a control. The sessile minimum inhibitory concentration
(SMIC50) is defined as the antifungal concentration at which a 50% decrease in
absorbance was detected in comparison with the control sample. All experiment pro-
cedures were performed three times for statistical analysis.
The SMIC50 results for C. albicans, C. krusei and C. glabarata treated with PMJ
for 10, 20 and 30 s are plotted in Fig. 16.6. Significant reductions of SMIC50 are
observed for all plasma treated Candida biofilms. SMIC50 ranges from 1 to 16 mg/
ml for fluconazole, 0.1250.25 mg/ml for amphotericin B and 0.0150.5 mg/ml for
caspofungin after a 10 s plasma treatment. These values are reduced to 1, 0.125
and 0.015 mg/ml, respectively, after 20 and 30 s treatments.
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 209
200
BMU 04801 BMU 05137 BMU 00271
SC5314 BMU 00279 BMU 04388
BMU 02971 BMU 05102 BMU 01689
150 BMU 03213
100
50
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
d 4
C. albicans e f
C. krusei C. glabrata
Amphotericin B (g/ml)
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
g 2.0
C. albicans h C. krusei i C. glabrata
1.6 BMU 04801 BMU 05137 BMU 00271
Caspofungin (g/ml)
0.8
0.4
0.0
0 10 20 30 0 10 20 30 0 10 20 30
Time (s)
Fig. 16.6 SMIC50 of (a)(c) fluconazole, (d)(f) amphotericin B and (g)(i) caspofungin for the
biofilms treated for 10, 20 and 30 s
350 350
OH (A-X)
O 844
O 777
x50
300 300
He 706
Emission Intensity (A.U.)
He 667
Emission Intensity (A.U.)
He 587
x250
He 501
250 250
Ha 656
200 200
He 728
150 150
300 305 310 315
100
He 447
100
He 492
7 5
32 32
He 471
C
50
u
C
0
0
300 350 400 450 500 550 600 600 650 700 750 800 850
Wavelength (nm) Wavelength (nm)
Fig. 16.7 End-on optical emission spectrum of He/O2 PMJ in air (flow rate: 2.5 slm; current:
35 mA)
(Acton 2750) equipped with a 1,800 groove/mm grating. The dispersed emission
spectra were recorded by an intensified CCD camera (Roper Scientific I-MAX-1024)
in the exit plane of the spectrometer. The light was focused into one end of the fiber
optics cable via a quartz convex lens. Figure 16.7 shows the end-on spectra of He/O2
PMJ operated in air in the UV to near IR region. Major peaks are mostly from helium
emissions, while strong atomic oxygen emissions at 777 and 844 nm are also
observed. Aside from direct electron impact excitation and dissociative excitation,
helium metastables must have participated in the excitation of oxygen from the car-
rying gas as well as from the air. Water from the surrounding air were disassociated
and excited. Emissions from both OH (A-X) band in 306309 nm and Ha at 656 nm,
although very weak, were observed (insets in Fig. 16.7). Copper emissions were also
observed in the UV region due to the choice of the electrode material.
Free radicals are atoms, molecules or ions with unpaired electrons. They are
highly chemically reactive and can therefore react with biological material. They
may be produced directly in the plasma or at the plasma-liquid interface of the sys-
tem. We believe hydroxyl radical (OH) and singlet molecular oxygen (1O2), are of
particular importance in our system. Both OH (oxidation potential: 2.8 eV) and 1O2
(oxidation potential: 0.98 eV) tend to attack chitin and polysaccharides on cell wall
as well as unsaturated fatty acids on cell membrane. Their presence can compro-
mise the function of cell wall and membrane lipids and cause the transportation of
ions and polar compounds into the cell [24].
Electron spin resonance (ESR) spectroscopy is a direct method for detecting species
which possess an unpaired electron, whose spin states are split in an external magnetic
field. Upon application of a magnetic field of the resonance frequency between the two
states, a transition is induced, which is signaled by an absorption peak on the spectrum
of the magnetic field. As both radicals mentioned above (in particular OH) are of rather
short life-span and are therefore difficult to be detected directly in ESR spectrum, spin
trapping was applied to facilitate the detection by reacting short-lived radicals with a
spin trap reagents. As a result, persistent aminoxyl spin adduct radicals are produced,
which are of longer life-span and are easier to be detected.
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 211
a 1500 b 2000
DMPO-OH TEMPO
750 1000
0 0
Fig. 16.8 Electron spin resonance spectra of (a) DMPO-OH (spin adduct of OH) and (b) TEMPO
(spin adduct of 1O2)
16.6 Discussions
Non-thermal plasma can effectively inactivate the plantonic cells of Candida spe-
cies (whether antifungal resistant of not) on Petri dish. We believe that in the treated
area, both charged particles and reactive species (short lived and long lived) partici-
pate in the inactivation process. In the untreated area, however, only longer lived
reactive species (such as O3) can contribute to the inactivation. Helium metastable
(~20 eV) has a longer life time, can essentially transport laterally to the untreated
area and interact with oxygen in air via stepwise excitation to create antifungal
212 W. Zhu et al.
species on-site. Nevertheless, Candida species in the untreated area can be inactivated
to the extent as in the treated area, but a bigger dosage (longer treatment time) seems
to be necessary. When the treatment was done in water, the Candida species were
not bound to agar as they were on Petri dishes but can move rather freely. They can
be brought to a close vicinity of the plasma (which was sustained in a quasi-steady
gas cavity in water). Furthermore, radicals with strong oxidative capability (such as
OH and 1O2) were generated in water. Both of these facilitate a fast inactivation of
Candida species.
Biofilms are structured microbial communities that are found in many places
(such as in catheters and on implanted artificial joints). They are resistant to both
host defense and commonly used antifungal drugs. The results here on ten Candida
strains (isolated from different sources) only show first evidence of the fast and
effective treatment of Candida biofilms and the change of their antifungal suscepti-
bility. The results were confirmed by cell viability test (XTT assay) as well as SEM.
The fact that biofilms were completely inactivated within 60 s in air while the
inactivation of planktonic fungi in the untreated area on Petri dish took 4 min may
seem that biofilms are easier to inactivate. However, one has to note that the treat-
ment of the planktonic fungi in air was performed on Petri dish in a 2 2 cm2 area.
The percentage of inactivation in the treated area is similar to that in the well of a
microtiter plate, which has a diameter of 6.86 mm. We believe that charged particles
and reactive species generated in the plasma are responsible for the inactivation.
Some of the radicals (such as OH) may not be produced directly in the plasma but
rather at the plasma surface interface (given surface water accumulation from sur-
rounding air). O2 must have been produced in the system. Evidence [25] has shown
that their conjugate acid (HOO) can peroxidize the fatty acid on cell membrane, at
pH lower than a critical value of 4.7. PH of our liquid system was found to change
from 7.3 only to approximately 7. O2 converts to OH in the system via a process
called Haber-Weiss reaction (O2 + H2O2 OH + OH + O2). This reaction is
accelerated with transition metal copper (electrode used in the device) as the cata-
lyst. Cu+/Cu2+ were detected in water by HPLC [26]. 1O2 is believed to be directly
produced in the plasma and subsequently transferred to the surface. Its lifetime in
air is in tens of minutes but is reduced to seconds in liquid. Nevertheless, oxidative
damages can be caused by these reactive oxygen species directly or indirectly, lead-
ing to impediment of ion transition. Furthermore, the oxidative molecules can com-
bine with DNA, causing additional cell damage [27].
In conclusion, atmospheric pressure He/O2 cold plasma microjet presents a new
approach to effectively inactivate C. albicans, C. glabara and C. krusei in plantonic
phase in air and in water as well as the biofilms induced by these strains (isolated
from different sources). However, issues concerning safety in operation, the function
of other killing agents in the plasma or at the plasma-surface interface, toxicity
study, their effect on healthy cells need to be further investigated in the future.
Acknowledgements Work supported in part by Bioelectrics Inc. (U.S.A.), the Peking University
Biomed-X Foundation and China International Science and Technology Cooperation (2008KR1330
Cold Plasma induced biological effect and its clinical application studies)
16 Inactivation of Candida Strains in Planktonic and Biofilm Forms 213
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microjet. Plasma Processes Polym, Online: DOI: 10.1002/ppap.201100041
27. Davies MJ (2003) Singlet oxygen-mediated damage to proteins and its consequences. Biochem
Biophys Res Commun 305:761770
Chapter 17
Non-thermal Atmospheric Plasma Treatment
for Deactivation of Oral Bacteria and
Improvement of Dental Composite Restoration
Abstract This paper reviews our recent research results of using non-thermal
atmospheric plasmas for oral bacterial deactivation and for composite restoration
improvement. Oral bacteria of Streptococcus mutans (S. mutans) and Lactobacillus
acidophilus (L. acidophilus) with an initial bacterial population density between
1.0 108 and 5.0 108 cfu/ml were seeded on various media and their survivability
with plasma exposure was examined. The plasma exposure time for a 99.9999%
cell reduction was less than 15 s for S. mutans and within 5 min for L. acidophilus.
To evaluate the dentin/composite interfacial bonding, extracted unerupted human
third molars were used by removing the crowns and etching the exposed dentin
surfaces with 35% phosphoric acid gel. After dental composite application and
light curing, the teeth were then sectioned into micro-bars as the specimens for
microtensile test. Student Newman Keuls (SNK) tests showed that the bonding
strength of the composite restoration to peripheral dentin was significantly
increased (by 64%) after 30 s plasma treatment of the dentin surfaces. These find-
ings indicated that non-thermal atmospheric plasma technology is very promising
for dental clinical applications.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 215
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_17, Springer Science+Business Media B.V. 2012
216 Q.S. Yu et al.
17.1 Experimental
An atmospheric cold plasma brush was used in this study. The detail of this
plasma source was reported previously [22, 23]. A MKS mass flow controller
17 Non-thermal Atmospheric Plasma Treatment for Deactivation of Oral Bacteria 217
Fig. 17.1 Optical emission spectrum of Ar atmospheric plasmas. Plasma conditions were 1,500
sccm Ar flow and 15 W DC power input
(MKS Instruments, Andover, MA, USA) was used to control argon gas flow rate.
The discharge was ignited and sustained by a DC power supply (Pd 1556c, Power
Design Inc. New York, NY, USA). With a relatively high gas flow rate, the plasma
discharge formed inside the chamber could be blown out of the chamber to form a
brush-shaped low temperature plasma jet, i.e., a cold atmospheric plasma brush.
This plasma source can be operated under very low electrical power (as low as a few
watts), and as a result very low plasma temperature can be achieved. The gas phase
temperatures of such an argon atmospheric plasma, measured using an infrared
camera combined with a thermocouple thermometer range from 30C to 65C, with
the corresponding argon gas flow rate varying from 500 sccm (standard cubic
centimeter per minute, a volumetric flow rate at 273 K under 1 atm) to 3,500 sccm
and input power from 5 to 15 W.
The optical emission spectrum (OES) of the Ar atmospheric plasmas is shown in
Fig. 17.1. Besides Ar emission lines, N2 emission lines and one O emission line
(777.2 nm) were observed due to the interaction of the Ar plasmas with ambient air.
Glass slide was used as smooth solid surface similar to the intact tooth surface and
PTFE film was a polymer surface carrier. Tryptic Soy Agar and Tryptic Soy Broth
(Difco Bacto, Detroit, MI, USA), Standard Methods Agar (Becton Dickson,
Cockeysville, MD), and Lactobacillus MRS Agar (MRSA) and MRS Broth (MRSB)
(Difco Bacto, Detroit, MI, USA) were used. The detailed experimental procedures
were reported elsewhere [30].
Fig. 17.2 Plasma sterilization effect on (a) S. mutans and (b) L. acidophilus seeded on different
support media. Plasma conditions were 2,000 sccm Ar flow rate and 10 W DC power input [30]
Fig. 17.3 SEM images of S. mutans and L. acidophilus cells without plasma treatment and after
plasma exposure for various time periods. The support media were glass slides and plasma condi-
tions were 2,000 sccm argon flow rate and 10 W DC power input [30]
Fig. 17.4 The change in absorbance (Abs.) intensity of intracellular protein and DNA leakage
from (a) S. mutans and (b) L. acidophilus with plasma treatment time. Plasma conditions were
2,000 sccm argon flow rate and 10 W DC power input [30]
proteins and DNAs. For both bacterial samples, a longer plasma exposure showed
continuous leakage of intracellular proteins and DNAs, in spite of leveling off of the
peak intensity of absorbency at wavelengths of 260 and 280 nm. These data suggest
that there were a large amount of protein and/or nucleic acids that were released into
the supernatant immediately after plasma exposure, which usually occurs when the
cell membranes are damaged as observed by SEM images shown in Fig. 17.3.
50
30
20
10
Completely
Penetrated
0
0 5 10 15 20
Plasma Treatment Time (second)
Fig. 17.5 Water contact angle change of dentin surfaces with plasma treatment time
Fig. 17.6 FTIR spectra of demineralized dentin before and after plasma treatment [33]
were two major changes in the representative spectrum of dentin surface after
plasma treatment. First, a new shoulder peak around 1,760 cm1 (in the oval)
associated with carbonyl stretch was found. This new peak has been confirmed by
subtraction line between the FTIR spectra of plasma treated and untreated dentin.
Second, an amide II shift of ~10 cm1 was observed (1,543 cm1 before to 1,533 cm1
after), which might indicate the secondary structural changes of dentin collagen
after plasma treatment. These chemical changes of the collagen fibrils may allow
more interactions with the adhesive resins applied subsequently.
After acid etching, the polished dentin surfaces were exposed to non-thermal
argon plasmas for predetermined time period as pictorially shown in Fig. 17.7. The
dental adhesive and dental composite resins were subsequently applied and light
cured to the dentin surfaces as instructed. Microbar test specimens were then pre-
pared with a cross-section of ~1.0 1.0 mm for tensile testing [33, 35]. The mean
17 Non-thermal Atmospheric Plasma Treatment for Deactivation of Oral Bacteria 223
cross sectional areas of each test specimen between treatments were from 0.87 to
0.95 mm2 as measured using a digital caliper and no difference was detected among
the treatments of all groups by one-way ANOVA (p = 0.38). Typically, 2025 micro-
bar test specimens were prepared from one extacted tooth. Among these 2025
microbars, about 46 microbars obtained from the tooth center position were desig-
nated as inner dentin (I) and the rest 1520 microbars were designated as peripheral
dentin (P). The bars were adhered to a universal testing system TAHD Plus (Stable
Micro System Ltd, Golalming, Surrey GU7 1YL, UK) via cyanoacrylate resin (Zapit,
Corona, CA, USA) and subjected to tensile testing with strain rate of 0.5 mm/min.
No specimens from the peripheral dentin failed prematurely before testing.
Figures 17.8 and 17.9 show the statistical comparison of ultimate tensile strength
and modulus data obtained with test specimens prepared from plasma treated dentin
and the untreated controls. Statistically significant differences in tensile strength
between all specimens using Student-Newman-Keuls (SNK) method were observed.
A significant difference was found between the peripheral dentin that was plasma
treated for 30 s and all the other treatments. As the plasma treatment time was
increased beyond 30 s the tensile strength decreased. After100 s plasma treatment,
the tensile strength of the microbar specimens was increased as compared to the
controls, but not significantly. After 300 s plasma treatment, tensile strength of the
specimens was similar to the control specimens, with a larger variance.
The modulus for the 300 s plasma treatment specimens was also reduced
(Fig. 17.9). Modulus data showed a trend of lower modulus for inner dentin as com-
pared to peripheral dentin. The 30 s plasma treatment increased the modulus of the
interface bonding when compared to the controls; however increasing treatment
time did not further increase the modulus. Inner dentin specimens for 100 and 300 s
plasma trials were not successfully tested. Plasma treatment of inner dentin for 30 s
does not affect the mechanical properties as seen in Figs. 17.8 and 17.9.
The stronger interface bonding obtained with plasma treated dentin can be also
attributed to the new surface functionalities and thus modified surface characteristics
introduced through plasma treatment. Plasma treatment would introduce residue free
224 Q.S. Yu et al.
80.00
60.00
b
50.00
b,c b,c
40.00
30.00 c,d
d
20.00
10.00
0.00
0s P 30s P 100s P 300s P 0s I 30s I
Plasma Treatment Time/Dentin Location
Fig. 17.8 Statistical comparison of ultimate tensile strength obtained with test specimens
prepared from plasma treated dentin and the untreated controls (0s P and 0s I). P Peripheral
dentin, I Inner dentin. Different letters in the plot indicate statistically significant differences
(a = 0.05) [33]
1000 a
Interface Modulus (GPa)
800
b
b,c
600 c,d
d c,d
400
200
0
0s P 30s P 100s P 300s P 0s I 30s I
Plasma Treatment Time/Dentin Location
Fig. 17.9 Comparison of tensile modulus obtained with test specimens prepared from plasma
treated dentin and the untreated controls (0s P and 0s I). P Peripheral dentin, I Inner dentin.
Different letters in the plot indicate statistically significant differences (a = 0.05) [33]
C-C
1030
Untreated collagen
Fig. 17.10 FTIR spectra of plasma treated collagen and the untreated controls with and without
applying HEMA
after, the dentin collagens were washed thoroughly with water for three times. FTIR
results (Fig. 17.10) show that HEMA was totally washed away from the untreated
dentin collagens. In contrast, in the plasma pretreated dentin collagens, the spectrum
clearly indicates the presence of HEMA as evident from the observed bands at 1,716
(C=O stretching), 1,158 (OC), 1,078 (COC), 1,030 (CC), respectively. It is also
noticed that HEMA monomers in the plasma pretreated collagens have polymerized.
This is based on the wavenumber shift of the OC band (at 1,158 cm1) in the pre-
treated dentin collagens. The position of this band in the HEMA monomer is located
at 1,162 cm1, shifted to lower wave-numbers in poly (HEMA). The results indicate
that the plasma introduced functionalities on dentin surfaces could initiate polymer-
ization of adhesive resin monomers and consequently form robust chemical bonding
at the adhesive/dentin interface. Compared with micromechanical bonding, chemical
bonding is much stronger, more stable, and durable. As a result, well improved inter-
face bonding strength was obtained (Fig. 17.8).
SEM images of the fracture surfaces generally showed that more composite
remained on plasma treated dentin surfaces when compared with the untreated con-
trols [33]. More specimens cohesively failed in the composite for plasma treated
specimens compared to the controls, except for the specimens prepared from 300 s
plasma treated dentin specimens. The control specimens had adhesive or mixed
failures more frequently than the plasma treated specimens. The large amount of
composite/adhesive observed on plasma treated dentin surfaces implies the dentin
adhesive interface is stronger than the bulk composite. These trends were also
observed with the test specimens that gave higher tensile strength. Plasma treated
226 Q.S. Yu et al.
specimens cohesively failed within the composite more frequently than the control
specimens which also implies a stronger interface. These experimental results
demonstrated that a proper plasma treatment of dentin could increase the bonding
strength of dental adhesives to dentin surfaces. It was also found that regional
variability and plasma modification duration affected the interface bonding strength
as measured using tensile test. An effective plasma treatment time was around 30 s
as observed from the mechanical testing data. This short plasma treatment is desired
in dental clinical applications because it will allow a quick dental composite restora-
tion. On the other hands, a prolonged plasma treatment could cause collagen fibers
to degrade and as a result lead to a weak interface.
17.4 Conclusions
Our experimental results showed that atmospheric plasma treatment was very
effective in rapid cleaning/disinfecting caries-causing oral bacteria. The results
obtained from this study showed the prospect of utilizing non-thermal plasma tech-
nology for dentin surface treatment to increase the chemical interactions of dentin
surfaces with dental adhesives, to introduce chemical bonding at the interface, and
thus to improve the interface bonding of composite restorations. It was demon-
strated that, under appropriate plasma conditions, plasma treatment of dentin could
increase the bonding strength of dental adhesives to dentin surfaces. The findings
from this research indicated that non-thermal atmospheric plasmas could be a
promising technique in dentistry for many clinical applications, e.g., oral bacterial
disinfection, caries early prevention, and improved composite restoration.
Acknowledgements The research work was supported in part by US National Science Foundation
(NSF) under contract of NSF-CBET-0730505 and US National Institute of Health (NIH) with
grant numbers of 1R43DE019041-01 and 2R44DE019041-02.
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Part III
Plasma-Based UV Sterilization
Chapter 18
Features of the Sterilization by VUV/UV
Irradiation of Low-Pressure Discharge Plasma
Vyacheslav V. Tsiolko
For the first time, plasma use for inactivation of microorganisms was proposed by
Menashi in 1968 in patent [1]. Sterilization of internal surfaces of glass or plastic
containers was performed by means of pulsed corona discharge in argon at atmo-
spheric pressure. It has been shown that the sterilization of spores with surface
density up to 6 105 spores/cm2 is achieved in a time of less than 0.1 s. Subsequently,
this author together with Ashman proposed usage of RF low pressure discharge on
chlorine, bromine and iodine containing gases for the surface sterilization [2]. In
both patents, sterilized items were placed immediately in the region of the discharge
plasma generation. In patent [3], another approach to design of the device for
sterilization was used: items to be processed and location of the plasma generation
were spatially separated along dielectric working chamber, and the sterilization was
performed by flowing afterglow argon plasma from low pressure capacitive RF
discharge. Subsequent patents proposed usage of different device designs, discharge
types and plasma generating media for the plasma sterilization. In patent [4] it was
proposed to use the plasma excited by ultra-short laser beam for sterilization of
internal surfaces of containers. It has been also shown that the use of low-pressure
capacitive RF discharge plasma in oxygen provides sterilizations of B. subtilis
spores on the surface of packed instruments for about 60 min [5]. Plasma of
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 231
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_18, Springer Science+Business Media B.V. 2012
232 V.V. Tsiolko
8
10
7 7 -2
10 10 cm
6 -2
Number of survivors
10
6 10 cm
5 -2
5 10 cm
10 4 -2
10 cm
4
10
3
10
2
10
1
10
0
10
0 200 400 600 800 1000
Sterilization time, s
Fig. 18.1 Survival curves for B. subtilis spores at sterilization by the hollow cathode discharge
plasma in air for test objects with different mean surface spore density values. Each point at the
graphs represents averaged results of counting for 612 test objects (24 individual sets). Power
density 0.01 W/cm3, P = 10.6 Pa (in part according to [16])
in three-phase survival curves. While at the first phase inactivation rate is determined
by DNA damage of the upper spore layer by VUV/UV irradiation, kinetics of the
second phase is determined by slower erosion processes (due to both etching by the
active plasma species, and VUV/UV photodesorption). The third phase happens
only when VUV/UV irradiation gets an access to the lower spore layers due to
removal of the upper ones. It is obvious that the situation, which occurs in actual
plasma sterilization, is essentially more complicated. Particularly, as it was shown
in [15], ion flows from the plasma may essentially accelerate spore erosion caused
by electrically neutral active plasma species. Thus, in general case, following from
common survival curves it is very difficult to separate influence of particular
plasma factors on the sterilization efficiency. Influence of the surface density of
B. subtilis spores at the test objects on a behavior of survival curves is demonstrated
in Fig. 18.1 (in part after [16]).
In these experiments, metal Petri dishes with bottom surface of 10 cm2 were
used as test objects. During the preparation of the test objects, Petri dishes were
at first treated in low pressure argon discharge plasma for surface wettability
improvement, and then the spore water suspension was deposited homogeneously
onto the dishes bottom (Initial population of the spores was varied in a range
105109). Quality of deposited spore layers at the surface of Petri dishes was mon-
itored by means of scanning electron microscope (SEM). One can see from
Fig. 18.1 that in the range of initial mean surface spores concentration 104106
spores/cm2, survival curves posses practically the same behavior (especially in the
second phase). As it was shown in [1719], in this case the main role is performed
by VUV/UV plasma irradiation. But increase of the spore concentration up to 107
spores/cm2 leads to drastic alteration number of survived spores at the same
sterilization times grows by more than one order of magnitude. Results of SEM
investigation of the test objects, in turn, have shown that, whereas at variation of
initial spore surface density from 104 to 106 spores/cm2, the spores are located in
a single layer practically everywhere at the test objects, then subsequent increase
of the spore surface density up to 107 spores/cm2 leads to clumping and occlusion
of the spores at the test objects surface. Thus, it is obvious that growth of the
number of survived spores at the increase of their surface density up to 107 spores/
cm2 is due to diminishing of VUV/UV irradiation action caused by its absorption
by upper layers of stacked spores. Due to that, in this case the spore inactivation
efficacy will be determined not only by VUV/UV irradiation action, but by slower
erosion processes as well.
Behavior of the spore survival curves at small initial surface density of the spores
(104106 spores/cm2) at the test objects also enables making a conclusion about the
origin of tailing of the spore survival curves. Since the experiments described above
were accomplished with the same strain of B. subtilis spores, and main role in the
inactivation is performed only by VUV/UV irradiation (that is, only one inactiva-
tion mechanism works in this case), then the most probable reason of biphase
character of the survival curves may be due to presence of two subpopulations of
spores with different resistances (as in case described in [13]).
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 235
20
0
40 80 120 160 200 240 280
Wavelength, nm
of fact that DNA extinction coefficient reaches its maximum values. In general, the
authors state that in each particular case the behavior of inactivation action spectrum
is determined by three factors: absorption of the VUV/UV radiation by outer layers
of the spores, the efficiency of DNA mortal damage production, and peculiarities of
repair mechanisms for each strain of the spores.
In turn, shape of VUV/UV irradiation spectrum of plasma in wavelength range
responsible for inactivation (that is, below 300 nm) is also a complicated func-
tion of several parameters: type of gas or gas mixture, the pressure, and the type
of plasma used for inactivation direct or flowing afterglow one. Usage of
gas with known electron transitions gives us principal possibility to obtain
required radiation spectrum shape. Particularly, when using pure gases/vapors
one can expect that: (1) in oxygen or water vapor, main contribution to VUV/UV
radiation of the plasma occurs due to emission of bands of the second negative
system (SNS) O2+ (A2Pu - X2Pg) (194300 nm) and Schumann-Runge system
(SRS) O2 (B3Su - X3Sg), ((244300 nm), (2) in nitrogen the radiation is repre-
sented by bands of Lyman-Birge-Hopfield system N2, (a1Pg - X1Sg+) (140
260 nm, with radiation intensity maximum in VUV branch) and
Lyman-Birge-Hopfield system N2, a1Pg - X1Sg+) (170200 nm), (3) in hydrogen
or NH3 radiation of H2 continuum in wavelength range 170300 nm (with max-
imum at about 200 nm) occurs. However, the situation is complicated at the use
of gas mixtures. Particularly, in case of N2 and O2 mixture, the main contribution
to UV radiation is provided by the bands of g (A2+ X2) and/or b (B2P X2P )
systems NO, at that their relative contribution to overall UV irradiation depends
both on concentration ratio of gas mixture components, as well as on nature of
the plasma which generates the species (direct or flowing afterglow). (It
should be noted that any pure gas actually contains admixture of ambient air
coming from the walls of gas feeding system and chamber. As well see below,
this admixture can also influence VUV/UV radiation spectrum). Such difference
of VUV/UV radiation spectrum of gas mixture plasma from spectra produced by
plasma of the mixture components is explained by fact that in case of gas mixture
essential changes are possessed by component content of the plasma species,
electron energy distribution function (which determines excitation efficacy of
molecule electron levels), and in addition to that, new channels of generation of
VUV/UV quanta occur.
In any case, one can state that spectrum distribution of VUV/UV plasma radia-
tion in a range below 300 nm is very uneven. Since there is a difference in inactiva-
tion action provided by the plasma irradiation at each particular wavelength, in
comparison of inactivation efficacy for different VUV/UV spectrum shapes one
should take into consideration germicidal portion of the emission [22, 23]. In other
words, one should weigh inactivation action at each wavelength. Due to that,
actual, that is weighed inactivation fluence rate F for each type of the spectrum is
presented by the equation
I (l )G (l )dl ,
300
F=
l min
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 237
Prior to proceeding with particular results of the researches, let us consider briefly
principal difference of features of flowing afterglow and direct plasma from the
viewpoint of generating VUV/UV radiation.
Generation of radiation in the plasma may occur by two ways: either due to a
direct excitation of certain electron level of plasma particle by the electron hit, or
at interaction of species formed in result of processes of dissociation and excitation
of gas medium particles. Since both in the first and the second case, the primary
role is performed by plasma electrons, efficiency of generation of VUV/UV radia-
tion is in any case determined by an appearance of plasma electron energy distribu-
tion function, and is mainly defined by the quantity of fast electrons with energies
exceeding thresholds of dissociation, excitation and ionization. In case of flowing
afterglow plasma, with moving away from the generation zone plasma, electrons
are cooled by collisions with ambient gas molecules (that is, quantity of fast
electrons decreases), which results in the decrease of generation efficacy of UV
(first of all, VUV) quanta. Besides, a component contents of the plasma species
also changes, which influences the appearance of UV radiation spectrum. Obviously,
the actual behavior is somewhat more complicated due to a big quantity of elemen-
tary processes occurring in the plasma. On the other side, when the plasma moves
away from the location of its generation, the temperature of electrically neutral
plasma species (which defines the temperature of processed items) also decreases
due to collisions. Thus one can see that the use of direct plasma for inactivation
enables more efficient VUV/UV irradiation, but at the same time, probability of
overheating processed items exists. And in flowing afterglow plasma, the tempera-
ture of the items is lower, but the irradiation efficacy decreases both due to the
lower intensity of UV radiation as a whole, and the depletion of VUV radiation
spectrum range.
238 V.V. Tsiolko
shown in Fig. 18.4. (R is a logarithm of ratio of initial number of the spores N0 = 106
to that of survived ones Ns).
As one can see from Fig. 18.4, the main role in the sterilization of B. atrophaeus
spores is performed by UV radiation in the wavelength range l 235300 nm,
whereas the influence of VUV radiation is less important. Although B. atrophaeus
is one of subspecies of B. subtilis, the obtained results are in certain contradiction
with the data of [30] where VUV irradiation performed dominating role in the inac-
tivation. Possible reasons for such discrepancy may be due to both the difference in
spectrum sensitivity of these spores, and different spectrum shapes of VUV/UV
irradiation broad continuum of H2 radiation in [30] and band-shaped radiation
spectrum of N2 and NO molecules in [31, 32], and probably to combination of these
factors.
The high frequency (200400 MHz) discharge on argon was used in [33]
for study of UV radiation role in the process of direct plasma sterilization of
B. atrophaeus (ATCC 9372) spores. (As well as in the works discussed above,
spores at the used test objects possessed monolayer arrangement). In that work, fil-
ters made of MgF2, SiO2 and Pyrex (lcut-off = 112, 190 and 330 nm respectively) were
used. Results of the experiments are given in Fig. 18.5.
One can see from the figure that sterilization of the spores is mainly performed
by VUV radiation in wavelength range 112190 nm. Spectrum identification for
VUV radiation in the range of 112180 nm was not accomplished, and its integral
intensity was measured by means of photomultiplier. Since cut-off wavelength of
filter is defined as the wavelength with 50% transmission, radiation intensity in
VUV range may be due to emission of argon resonance lines located near 105 and
107 nm. However, due to fact that experimental conditions did not allow direct
measurements of intensities of these lines, another approach was used. By the
method of absorption spectroscopy, the dependence of relative population density
of the metastable level 3P2 on argon pressure was determined. Since the authors had
to determine relative variations of the population density, rather than absolute ones,
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 241
R
(After [32]) 3
2 SiO2=300 nm
1
0
0 100 200 300
cut-off / nm
and respective populations of resonant and metastable levels of the 3p54s orbital
configuration are of the same order and vary similarly to the functions of the opera-
tion conditions, such substitution was justified. It has been also shown that, if the
sterilization efficiency increases along with argon pressure growth, then relative
population density of the metastable level 3P2 (and, respectively, radiation intensity
of lines at 105 and 107 nm) decreases. At the same time, integral radiation intensity
in the range of 112180 nm increases with the pressure growth. Thus, it is clear that
radiation of argon resonance lines at 105 and 107 nm does not contribute to spore
inactivation. Simultaneous measurements of radiation spectrum in the range of
200400 nm have indicated presence of radiation of NOg system, the 306.4 nm sys-
tem of OH and the 2nd N2 positive system, which gives evidence to presence of
admixtures (residual air, water vapor) in argon. Due to that, the authors have assumed
that the main contribution to VUV radiation in the range of 112190 nm is done by
242 V.V. Tsiolko
with oxygen practically coincide with each other. Curves obtained at the use of UV
radiation of mercury lamps and that of the discharge plasma on air are also close to
each other, however, they are located above the first set of the curves. Such essential
difference in behavior of the curves gives an undoubted evidence to the fact that
bactericidal features of UV radiation in 215300 nm wavelength range depend not
only on radiation dose absorbed by DNA (as it was observed in the case of mono-
chromatic radiation), but on shape of the radiation spectrum as well. Comparison of
spectra of radiation absorbed by DNA for the discharges on different gases shows
that maximum inactivation efficiency is provided by the discharges plasma with UV
radiation having maximum flux rate in 215230 nm range.
To determine the fact whether high inactivation efficacy by UV radiation with
maximum fluence rate in 215230 nm range is specific only for vegetative form of
microorganisms, or has more general character, authors of [30, 31] have done veri-
fication of this effect for the case of spores B. subtilis received from Scientific
Research Institute of Standardization and Control of Medical Biological Preparations
(Moscow, Russia). The experiments were performed at the setup, which was
described in details in [17]. Measurements of spectrum dependencies of the plasma
UV radiation in wavelength range of 200300 nm on the discharge glow time tg
were performed by means of spectrometer SL40-2-2048USB (SOLAR TII, Ltd)
with the spectral resolution 0.2 nm. At the measurements, the end of quartz wave-
guide of the spectrometer was located in a plane, which corresponded to placement
of Petri dishes with B. subtilis spores during medical-biological researches. The
discharge parameters were the same as in the case of inactivation of these spores in
[1719]: pressure of oxygen and ambient air was varied in a range of 416 Pa,
specific power in the discharge Wd in a range of 0.0025-0.0125 W/cm3. For correct
comparison of inactivation results with the use of UV radiation with different
spectrum shape, weighing of spectrum distributions of the intensity of UV radia-
tion from oxygen and air plasma was performed. Inactivation action spectra for
B. subtilis spores type RCF from [21] was used as weighing function. (It should
244 V.V. Tsiolko
a b
60 240
"Weighed" UV fluence rate Ew, a.u.
40 160
30 120
20 80
10 40
0 0
200 220 240 260 280 300 200 220 240 260 280 300
Wavelength, nm Wavelength, nm
Fig. 18.7 Spectral distributions of weighed fluence rate Ew of UV radiation from oxygen (a) and
air (b) discharge plasma. Pressure P = 15 Pa, Wd = 0.08 W/cm3
be noted that the use of the inactivation spectra, obtained in [21] for other spore
types, as weighing functions results in values of weighed UV fluence rate Ew
and fluence Fw of the radiation, which differ by no more than 2025%, as compared
to the values in case of RCF weighing function use). Figure 18.7a and b shows the
weighed spectrum fluence rate Ew distributions of UV radiation from oxygen and
air plasma.
As one can see from the analysis of these spectra, at oxygen use the main
contribution to UV radiation of the plasma occurs due to the emission of the second
negative system O2+ (A2Pu X2Pg) and Schumann-Runge system O2 (B3Su X3Sg),
and at the use of ambient air and nitrogen due to the emission of g system NO (A2S+
X2P). For both cases fluence Fw of UV radiation practically linearly grows with tg
and Fw value for the discharge plasma in ambient air considerably exceeds UV
fluence for the discharges in oxygen in the whole range of tg variation. The experi-
ments have also shown that at all gas pressures, the values UV fluence value grows
up linearly with the increase of discharge power Wd. In Fig. 18.8 the numbers of
survivors B. subtilis spores vs UV weighed fluence Fw from discharge on oxygen
and ambient air plasma are presented.
These survival curves were obtained by converting dependencies of the number
of survivors spores on the sterilization time ts (see insert in Fig. 18.8) with the use
of respective dependencies of fluence Fw on tg. One can see from Fig. 18.8 that
B. subtilis spores sterilization in case of use of UV irradiation from oxygen plasma
is reached at fluence Fw approximately five times less than in case of air plasma.
The observed large difference in the behavior of survival curves for inactivation
of the spores by UV from oxygen plasma on one side, and from air one on another
side, unambiguously shows that bactericidal features of UV radiation in 200300 nm
range depend not only on the UV fluence, but also on the shape of radiation spec-
trum. And this effect is possibly inherent not only to the types of the microorgan-
isms described above. High efficiency of UV radiation having maximum in
wavelength range 200240 nm may be presumably due to: (1) difference in nature
of DNA damage caused by UV radiation in the mentioned wavelength range, as
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 245
7
10 10
7
6
6 10
10
Number of survivors
5
10
4
5 10
10
Number of survivors
3
10
2
4 10
10 1
10
0
3 10
10 a b
-1
10
2 0 100 200 300 400
10 Inactivation time ti, s
1
10
0
10
a b
-1
10
0,0 0,1 0,2 0,3 0,4 0,5
"Weighed" UV fluence Fw, a.u.
And only in [37], the effect of temperature of Petri dish with B. atrophaeus spores
on the efficiency of their inactivation by UV radiation (NOb system) from flowing
afterglow MW N2-O2 discharge plasma was studied in detail. During the experi-
ments, the Petri dish was tightly closed by CaF2 optical filter (lcut-off = 112 nm) to
prevent plasma active species entering. The temperature of the Petri dish could be
changed from 4C to 80C. It has been shown that inactivation rate increases mono-
tonically with the temperature of Petri dish only in the case when heat and UV
radiation are applied simultaneously. In the cases when Petri dishes were heated
before or after their exposure to plasma afterglow at low temperature, synergetic
effect was not observed. Authors explain the growth of UV spores inactivation rate
with the temperature increase by fact that the heat provides the energy required to
surmount the potential barrier(s) encountered as the chemical reaction, initiated by
photon excitation. The energy barrier corresponds to molecular (conformation)
rearrangements occurring after photoexcitation, as the reaction develops to reach
the final state creating the lethal damage to the spore DNA strands.
In a majority of accomplished researches on VUV/UV plasma irradiation inacti-
vation, the temperature of the test objects was not stabilized and grew up with expo-
sure time. Let us consider qualitatively a character of the discovered effect influence
in case of investigating VUV/UV inactivation of the spores by radiation with differ-
ent spectrum distribution of the irradiation fluence rate. From insert in Fig. 18.8 one
can see that the sterilization by UV irradiation of air plasma is achieved about 3 min
later than that by UV irradiation of O2 plasma. Temperature of exposed to plasma
Petri dishes grows up practically linearly in time with a rate of about 1.5C/min.
That is, at reaching sterilization, temperature values of the test objects differed very
slightly no more than by 45C, whereas in [37] considerable difference in a slope
of survival curves was observed at temperature difference of 2030C. Certain
increase of the inactivation efficacy in case of air plasma use could result just in
moving air survival curve toward oxygen one. That is, it would just somewhat
decrease influence of the difference in spectrum distributions of UV fluence rate of
oxygen and air plasmas on the inactivation efficacy, without influence on the
effect as a whole.
18.4 Conclusions
Although during the past decade intense researches of low-pressure plasma UVU/
UV microbial inactivation were performed, their results cause contradictory feeling.
On one side, many researches were accomplished on the generation of UVU/UV
radiation of different discharge plasmas (DC, RF, HF and MW types) in various gas
mixtures at the different pressure, and dependencies of VUV/UV inactivation effi-
cacy on radiation spectrum shape and other parameters were determined for differ-
ent types of microorganisms (vegetative, fungi, spores). On the other side, conditions
of particular researches differ considerably, which makes correct quantitative com-
parison of the obtained results practically impossible.
18 Features of the Sterilization by VUV/UV Irradiation of Low-Pressure 247
For solving the problem, one should standardize the conditions of conducting the
researches. Particularly, definite protocol is required for determining VUV/UV
spectral distribution of fluence rate and VUV/UV fluence given to the microorgan-
isms. (At present, published articles present only spectrum distributions of UVU/
UV radiation intensity in arbitrary units, usually measured at random points of the
space). Obviously, this task is more difficult than that for the case of determination
of the fluence in an apparatus containing monochromatic or broadband UV lamps
[23], but the efforts on development of such protocol will definitely justify them-
selves in the future.
Used test objects with the microorganisms should be also standardized. Variety of
used test objects (polymer foils, glass plates, glass and polymer Petri dishes, etc.)
may also cause difficulties in comparison of the results of different works. Particularly,
at the same discharge plasma parameters, VUV/UV fluence value will be different
for plane test objects and Petri dishes due to the difference in acceptance angle of the
irradiation. Use of test objects with different mass, made of materials with different
heat capacity may also have an effect on the results of the researches due to influence
of the object temperature on VUV/UV inactivation efficacy [37].
Errors in determining efficacy of the plasma VUV/UV inactivation may occur
when separate points of the survival curves are used instead of the complete curves,
due to nonlinearity of dependencies of the number of survived microorganisms on
VUV/UV irradiation fluence.
As a whole, one can say that taking these wishes into consideration at accom-
plishing the researches may not only clarify understanding the processes during
VUV/UV inactivation, but as well promote the use of this method in medical
practice.
Acknowledgments The author would like to offer thanks to Dr. V. Yu. Bazhenov for helpful
discussions and assistance, Dr. Z. Machala for the encouragement and critical reading of the
manuscript.
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Chapter 19
Applications of Excilamps in Microbiological
and Medical Investigations
Abstract In a course long-term and comparative studies it has been shown, that the
DBD XeBr-excilamps looks as a good choice for various microorganisms inactiva-
tion. The first data about bacteriophage inactivation by XeBr-excilamp has been
obtained. Radiant modules for industrial treatment on contaminated water have
been developed. The XeCl-excilamp for treatment of skin diseases has been created
and tested.
19.1 Introduction
Spontaneous radiation sources, such as the excimer and exciplex lamps (excilamps)
find wide applications for science and engineering, in particular in biology and
medicine [17]. The main reasons are the following:
The great part of excilamps light energy concentrates in UV or VUV spectral
range which depends on gas mixture in a bulb. Photons with energies 510 eV
(UV and vacuum UV spectral ranges) can initiate and support different chemi-
cal, physical, and biological processes. Other advantages of excilamp are their
simplicity in comparison with UV and VUV lasers, and have long lifetime.
In the current review the excilamps are described briefly, and the most part of text
is dedicated to applications of excilamps in biology and medicine.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 251
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_19, Springer Science+Business Media B.V. 2012
252 V.F. Tarasenko et al.
Fig. 19.1 Emission spectrum and general view of lighting of XeBr-excilamp excited by CD (left)
and DBD (right). The operating pressure values were 3 and 97 Torr, accordingly
19.3 Applications
Fig. 19.3 Action spectrum of UV inactivation on E. coli (1), DNA absorption spectrum (2) and
maxima of radiation bands of various excilamps and low pressure mercury lamp (LP Hg-lamp)
The peak intensity of B-X band of XeBr* molecule (282 nm) is seen to be at
about the same distance from the action spectrum maximum, the same as resonance
line of a LP Hg-lamp (Fig. 19.4). That is Dl1 ~ Dl2. This suggests that the both
lamps have comparable bactericidal effect. Note that we do not know any other
papers reporting on such direct comparison made. The model of XeBr-excilamp
(model XeBr_BD_P, High Current Electronics Institute SB RAS) with the follow-
ing parameters: discharge gap 0.8 cm, tube diameter 4.2 cm, UV radiant exitance of
3 mW/cm2, and a spectrum shown in Fig. 19.4, was used in the experiments.
Another lamp was the conventional germicidal LP Hg-lamp (TUV-15). In the
experiments, the lamp was specially furnished with a diaphragm in order to provide
the radiation doses comparable in values with the radiation doses of the DBD-driven
XeBr-excilamp. As a result, the Hg lamp provided radiant exitance of 2.5 mW/cm2.
The lamps radiant power was defined in absolute units by using a C8026 (Hamamatsu
Photonics KK) photodetector with the H8025-222 head. Just after the initiation, the
XeBr-excilamp achieved its mode, and the LP Hg-lamp needed 2.5 min for its initial
heating to provide stable luminous flux.
The object of study was the pure culture of Escherichia coli (strain K12 ATCC
25922) provided by the Scientific Research Institute of Balneology (Tomsk, Russia). Our
selection of colibacillus for study was determined by its belonging to the main species of
the enterobacterium group taken for sanitation of desinfection efficiency by UV-radiation.
Besides that, the E. coli has one of the most high resistance coefficients among enterobac-
teria group. The bacterial cultures were supported on beef-extract agar (BEA) and kept at
the temperature of 4C. In preliminary study, based on the method of multiple dilutions,
the optimal concentration of microbial dredge was found for experiments.
256 V.F. Tarasenko et al.
Fig. 19.4 Different spectral characteristics, essential to this investigation: 1 UV action spectra of
DNA, 2 the absorption spectrum of DNA, 3 emission spectrum of DBD-driven XeBr-excilamp,
4 resonance line of LP Hg-lamp [15]
Primary and secondary irradiation of E. coli via various exposure values were
carried out. Usually, the irradiation was made 5 cm distant from the lamp to 10 cm
Petri dish with contaminated surface. Thereby the heating effect of substrate was
minimized and values of irradiation were kept high. Thus, uniform illumination of
contaminated surface was additionally provided. The microorganisms, which
survived after irradiation by a dose leading to inactivation of 99.9% of E. coli, were
allocated in pure culture and subjected to reirradiation. The qualitative and quantita-
tive analysis and estimation of bacterial colonies morphology (size, form, consis-
tence, character of edge) were made. The results of the experiments are shown on
Fig. 19.5.
Thus, according to our hypothesis, both types of the light sources provide the
similar bactericidal effect due to their radiation in the spectral range where UV
action spectra of DNA have comparable values. As compared with traditional mer-
cury bactericidal lamps, the advantages of the modern excilamps, accentuating to
their bactericidal application are: (1) high photon flux qp, extracted from plasma
without self-absorption; (2) no elementary mercury in bulbs, which conforms with
ecology; (3) extraordinary geometric freedom of bulbs; (4) momentary launching
and full radiant power after ignition; (5) variable tuning of photon flux qp.
As we noted above for the fist time we have established bactericidal effect of
CD XeBr-excilamps due to their wideband spectra in 2002. Such a spectrum has
a short-wave tail from 230 to 282 nm (B-X band), which covers a half of the first
maximum of DNA absorption. Besides, the spectrum has a D-X band with a
19 Applications of Excilamps in Microbiological and Medical Investigations 257
Fig. 19.5 Inactivation of surface inoculated E. coli, performed with various UV-doses of XeBr-
excilamp () and LP Hg-lamp (o) irradiation. The first bacteria generation results are on the left,
and the second bacteria generation results are on the right [15]
maximum at 221 nm, which has a short-wave tail from 210 to 221 nm. It has been
shown that the microorganisms, which survived after the first irradiation by that
excilamp, kept their radiation susceptibility unchanged [15]. UV resistance pro-
tection of microorganisms under the action of wideband UVB irradiation is
explained by the fact that biological structure of bacteria bears very many induced
failures of biological structure, making it improbable to appear stable mutants to
UVB irradiation (the viewpoint validity is being considered in the reviews [16]).
At LP Hg-lamp ruled irradiation one might expect appearance of the greater
number of UV resistant mutants than at irradiation by a wideband radiation source.
Just in this respect maybe be interpreted the fact obtained in the present investiga-
tion. At irradiation of bacteria, survived after the first irradiation (their average
share was 0.5%), the survival curve obtained for LP Hg-lamp activated bacteria
has moves aside from the curve obtained for XeBr-irradiated bacteria. In other
words, resistance of E. coli, activated by the LP Hg-lamp, is higher in XeBr-
excilamp irradiation case.
In [17] the comparison of XeBr-, KrCl- and KrCl + KrBr-excilamps radiation
impact on 5 microbiological cultures (Escherichia coli (ATCC 25923),
Staphylococcus aureus (25923) and extracted from human skin representatives
p. Sarcina, p. Pseudomonas and p. Bacillus) have been presented.
Emission spectrums of DBD-driven excilamp under using are presented on
Fig. 19.6. Data about bactericidal efficiency is assembled to Table 19.1.
258 V.F. Tarasenko et al.
It is shown that surface irradiation dose which gives 99.9% of bactericidal effi-
ciency for different excilamp and for LP Hg-lamp have a comparable values (if we
take into the mind the data from [18]). In the second place the bactericide efficiency
are decrease in a row of lamps XeBr (282 nm) > KrCl_KrBr (222 and 206 nm) > KrCl
(222 nm).
In 20092010 we have studied the sensitivity of hospital infectious agents to UV
radiation of excilamp and LP Hg-lamp using the abovementioned cultivation and
irradiation methods [19].
The object of study was the pure culture of Escherichia coli (strain ATCC 501),
Klebsiella pneumonia (strain ATCC 2482), S. aureus (strain ATCC 209) and two
cultures selected from patients of Tomsk Savinich Hospital C. albicans and
P. aeruginosa. Suspension of daily cultures (with concentration 105 CFU/ml in vol-
ume 0,1 ml) was inoculated into a meat infusion agar. As the control a suspension
of daily cultures in concentration 103 CFU/ml was used.
19 Applications of Excilamps in Microbiological and Medical Investigations 259
Fig. 19.7 Sensitivity of test cultures to UV radiation of excilamp and LP Hg-lamp after 15-s time
exposure (and at the same UV exposure)
The results of our research are illustrated in Fig. 19.7. From this figure we notice
that: (1) XeBr-excilamp irradiation gives better germicidal effect for E. coli,
P. aeruginosa cultures; (2) S. aureus and C. albicans cultures have the same
UV-resistance for both light sources.
The low UV-sensitivity of K. pneumoniae could be explained by presence of
sheath (capsule) in their biological structure. This capsule absorbs a part of radia-
tion flux and decrease the DNA damage frequency.
Our recent results (2010) are dedicated to the sensitivity of MS2 bacteriophage
under UV radiation of LP Hg-lamp and XeBr-excilamp. The model XeBr_BD_P
and LP Hg-lamp (TUV-15) were used in the experiments. The object of study was
the MS2 bacteriophage (strain PH-1505), which cultivated on E. coli K 12 F + (strain
B-3254) after irradiation. Both cultures are from Russian industrial bank of micro-
organisms. UV sensitivity of bacteriophage has been determined by well-known
Gratia method [20]. The exposure value was equal to 45 J/m2. Virocide action of
radiation has been estimated by quantity of blank places on Petri dishes (see
Fig. 19.8).
The greater viroicidal influence on tested culture has been achieved by means of
XeBr-excilamp (Fig. 19.9). We think that it is related to damaging genetic block of
bacteriophage as well as protein shell too. Our results testifiers that the excilamps
could be use in antiviral applications.
Concluding this chapter, let us note that some microorganisms and cells possess
UVA/VIS repair mechanisms (photoreactivation) that substitute or dissociate thy-
mine dimers. Under these circumstances, excilamps as narrow-band emission
260 V.F. Tarasenko et al.
Fig. 19.9 Sensitivity of MS2 bacteriophage to UV radiation of XeBr excilamp and LP Hg-lamp
at the same UV doses (45 J/m2) compared with control
sources should be more efficient than wide-band MP Hg lamps. Sure, this problem
needs further studies to be done.
Fig. 19.10 Example of psoriasis curing by the XeCl excilamp (model BD_P, see previous chapter)
in Siberian State Medical University (BD_compact model, Optical Radiation Laboratory, Russia,
output window square 30 cm2, UV photon exitance of 40 mWcm2): before curing (left) and after
10 days at suberythermogenic doses treatment (right) [25]
19.4 Conclusion
Acknowledgements This work was supported in part by the Federal Target Program The scien-
tific and scientific-pedagogical personnel of Innovative Russia, State contract No. 02.740.11.0562.
Discussions with L.V. Lavrenteva, U. Kogelschatz, T. Oppenlender and technical assistance of
S.M. Avdeev, A.V. Gritzyta, M.V. Erofeev, D.V. Schitz, V.S. Skakun are gratefully acknowledged.
References
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flow through photoreactor. Chemosphere 30:17811790
3. Lomaev MI, Skakun VS, Sosnin EA, Tarasenko VF, Shitts DV, Erofeev MV (2003) Excilamps:
efficient sources of spontaneous UV and VUV radiation. Phys Usp 46:193210
4. Sosnin EA, Oppenlnder T, Tarasenko VF (2006) Applications of capacitive and barrier
discharge excilamps in photoscience. J Photochem Photobiol C Photochem Rev 7:145163
5. Lomaev MI, Sosnin EA, Tarasenko VF, Shits DV, Skakun VS, Erofeev MV, Lisenko AA
(2006) Capacitive and barrier discharge excilamps and their applications. Instrum Exp Tech
49:595616
6. Sosnin EA, Sokolova IV, Tarasenko VF (2008) Development and applications of novel UV and
VUV excimer and exciplex lamps for the experiments in photochemistry. In: Sanchez A,
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industrial applications. Plasma Chem Plasma Process 23:146
8. Sosnin EA, Erofeev MV, Lisenko AA, Tarasenko VF, Shits DV (2002) Study of the service
characteristics of a capacitive-discharge excilamp. J Opt Technol 69:509511
9. Avdeev SM, Sosnin A, Tarasenko VF (2010) Factors that limit the service life of sealed
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11. Sosnin EA, Lavrenteva LV, Yusupov MR, Masterova YV, Tarasenko VF (2002) Inactivation of
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pressure plasmas: review, analysis and prospects. IEEE Trans Plasma Sci 30:14091415
15. Avdeev SM, Sosnin EA, Velichevskaya KYu, Lavrenteva LV (2008) Comparative study of
UV radiation action of XeBr-excilamp and conventional low-pressure mercury lamp on bacte-
ria. Proc SPIE 6938:693813
16. Kalisvaart BF (2004) Re-use of wastewater: preventing the recovery of pathogens by using
medium-pressure UV lamp technology. Water Sci Technol 50:337344
17. Avdeev SM, Velichevskaya KYu, Sosnin EA, Tarsenko VF, Lavreteva LV (2008) Analysis of
germicidal action of UV radiation of excimer and exciplex lamps. Light Eng 16:3238
19 Applications of Excilamps in Microbiological and Medical Investigations 263
18. Guidance P (2004) Using of bactericidal UV radiation for air decontamination in a housing,
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Radiation source. Patent RU2 271590. Priority date 15 Mar 2004
Chapter 20
Xenon Iodide Exciplex Lamp as an Efficient
Source for the UV Surface Cleaning
and Water Decontamination
20.1 Introduction
M. Guivan (*)
Department of Quantum Electronics, Uzhgorod National University,
Pidgirna 46, 88000 Uzhgorod, Ukraine
e-mail: m_guivan@rambler.ru
H. Motomura M. Jinno
Department of Electrical and Electronic Engineering, Ehime University,
Matsuyama, Japan
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 265
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_20, Springer Science+Business Media B.V. 2012
266 M. Guivan et al.
20.2 Experimental
(c) home made power supply (PS2), based on fast high voltage transistor push-pull
switch HTS 31-01-GSM (BEHLKE) and providing unipolar pulses with
U = 03 kV, rising and falling times Dtrise = 60 ns and Dtfall = 40 ns respectively,
f = 10 kHz;
(d) home made power supply (PS3), based on HTS 81-06-GSM push-pull switch
(BEHLKE), providing unipolar pulses with U = 08 kV, controlled rising and
falling times down to 160 and 60 ns respectively, f = 1080 kHz.
The voltage U applied to the excilamp was measured using a high voltage
probe (Sony Tektronix P3000 or Tektronix P6015). The waveforms of current I
and charge Q were registered by means of 100 W resistor or low-inductance 3.3 nF
capacitor placed in series with the excilamp and monitored by digital oscilloscope
TDS 3034B (Tektronix). The input power Pin was determined from the Lissajous
UQ figures.
The radiation power of the excilamp in absolute units was measured by a UV
power meter C8026 (Hamamatsu Photonics K.K., Electron Tube Centre) equipped
with a calibrated H8025-254 sensor head. The correct determination of the total
radiant flux requires the measurement of the angular distribution of radiation
from the tested DBD excilamp, because it differs from a Lambert source or a thin
linear lamp in geometry in the effect of the outer electrode and the emission from
the ends of the lamp. The distance L between the middle of the lamp and the
sensor head must be about 10 times longer than the active length of the lamp l.
Since the emission from the lamp is isotropic around the axis, the total UV radi-
ant flux Pout from the lamp (in the range of the sensor head sensitivity) can be
represented by
where Pmeas(Q) is the specific UV power in mW/cm2 measured by the power meter
in a certain direction and Q is the angle between the lamp axis and the sensor head
position, L = 1 m when l = 10 cm. The lamp efficiency was determined as the ratio
between the total UV radiant flux Pout and the input Pin: h = (Pout / Pin ) 100%.
Emission spectra were measured by means of HR4000 (5 mm slit, 1,200 g/mm
grating, 200400 nm) and USB2000 (10 mm slit, 600 g/mm grating, 200850 nm)
spectrometers (Ocean Optics). The registration system was calibrated within the
200850 nm wavelength range by the standard xenon lamp L7810-02
(Hamamatsu). For measurements of the emission features in the VUV range
(<200 nm) the VM-502 0.2 m vacuum monochromator (Acton) equipped with an
ICCD camera (PI-MAX, Princeton Instruments) and a PMT R928 (Hamamatsu)
were used. Calibration was performed by means of a deuterium lamp L648M
(Hamamatsu).
Figure 20.1 shows the experimental arrangement for the UV treatment of B. sub-
tilis spores in the water flow reactor. A chamber with a water spread system was
made of stainless steel. The test water (1 l) was circulated by a tubing pump
(Masterflex, Cole Parmer Instrument Co.) at a constant speed of 5.4 l/min.
268 M. Guivan et al.
The spread system provided a uniform water layer with a width and thickness of 90
and ~0.5 mm, respectively. To evaluate the exponential decrease of survival rate (N/
N0), the UV dose (cumulative energy density) needed to reduce a culturable cell
number was measured by a CFU counting assay. The distance between the XeI*
excilamp and the water layer was 4 cm. The test water was irradiated from 1 to
30 min using this experimental setup.
The relative energy, i.e. the contribution of each emitting species i in the total
output, was calculated as the area ratio Si/S170-400 from the VUVUV spectrum,
corrected on the spectral sensitivity of the registration system. The effect of
atomic iodine lines in the total UV output was estimated taking into account the
measured line width of 0.120.14 nm (FWHM). About 85% of radiation was
concentrated in the germicidal region. We have revealed that the atomic iodine
emission rises faster with frequency in comparison to XeI*. For pulsed excitation
with short voltage rising and falling times, when the operating frequency changes
from 10 to 80 kHz, the XeI*(253 nm) intensity increases ~5-fold, whereas the
I*(206 nm) intensity 19 times, and the intensity ratio 206 nm/253 nm grows
almost to 4 times [7]. In other words, we propose that by operating at a higher
frequency it is possible to get the greatest intensity of atomic iodine emission
and, consequently, better sterilization action due to larger DNA absorption. Note
also that the VUV part can be increased by using a quartz tube with enhanced
transmittance for l < 200 nm, such as Suprasil 311, 312 or Heralux plus [ 8 ] .
It was important for us to compare how the irradiation spectra affect the B. subtilis
spores lethality.
20 Xenon Iodide Exciplex Lamp as an Efficient Source 269
20.3 Results
Representative examples of measured voltage U(t) and external total current I(t)
waveforms, as well as UQ loops for Xe/I2 = 13.3/0.04 kPa mixture under the differ-
ent excitation schemes are shown in Fig. 20.2. The temporal evolution of all the
internal electrical quantities (discharge current, voltage across the dielectric, volt-
age across the discharge gap, consumed energy per cycle, and instantaneous input
power) in the gap were calculated using the technique described in [9, 10] and will
be presented elsewhere.
When using AC excitation, in each half-cycle of the applied voltage U0, the cur-
rent waveform consists of a displacement current and sharp current peaks caused by
the discharge (Fig. 20.2a), with characteristics depending on the operating fre-
quency and applied voltage. The peak current did not exceed 55 mA and their dura-
tion was in the range 0.92 ms. As U0 increased, the first current pulse shifted ever
more to the left of the peak of the applied voltage and, at high amplitudes U0 ~ 3.7 kV,
it occurred at the phase of reverse applied voltage. Up to three current pulses (three
three mode) were observed in each half-period under the present experimental con-
ditions. Similar behavior for the AC driven DBDs has been reported before, for
example, for He [11], Kr/I2 [12], and CdBr2/(Ne, Ar, Kr, Xe, N2) [13] mixtures.
For pulsed excitation the peak current was in the range 0.40.8 A and strongly
depended on the Dtrise and Dtfall values. The duration of the current pulses (FWHM)
did not exceed 200 ns. The same peak current was observed when Dtrise Dtfall
(Fig. 20.2e), whereas the shorter voltage pulse edge provides the greater current
amplitude (Fig. 20.2c, g). Note that pulsed excitation provides peak input power
into the discharge about one order higher than AC.
The DBDs driven by unipolar pulses show different electrical behaviour from
bipolar (Fig. 20.2e, g). The power input proceeds only during the primary discharge.
One part of the injected energy from the external circuit directly supports the pri-
mary discharge; the rest is stored by memory charges, to be released later to energize
the secondary discharge shortly after the pulse falling edge. The number of current
pulses for pulsed bipolar operation is completely controlled by the Dtrise and Dtfall.
The UQ loops can be efficiently used not only for the determination of the con-
sumed energy by the discharge, but also for the calculation of the lamp capacitance
Clamp for periods corresponding to the different discharge phases [9, 10]. Besides
that, from comparison of the calculated Clamp for cylindrical capacitor with known
geometric parameters and the slope dQ/dU in measured UQ loops, it is possible to
determine the conductivity of the discharge gap in the given time period [9]. In our
case we calculated Clamp ~ 7.5 pF, whereas the slope in Fig. 20.2b gives 1825 pF for
Off periods when the discharge current is negligible. The shape of the UQ loop
shows that at a given pressure under AC excitation discharge current flows during
270 M. Guivan et al.
3 60
2 40
50
Charge (nC)
Current (mA)
1 20
Voltage (kV)
0 0 0
1 20
2 40 50
3 60
100
0 10 20 30 40 50 2 0 2
Time (ms) Voltage (kV)
c Xe/I2=13.3/0.04 kPa, f = 60 kHz d 3.8 kV pp, 60 kHz, Pinput=17.62 W
2 0.4 100
1 0.2 50
Charge (nC)
Voltage (kV)
Current (A)
0 0.0 0
1 0.2
50
2 0.4
100
0 5 10 15 20 2 1 0 1 2
Time (ms) Voltage (kV)
e 1
Xe/I2=13.3/0.04 kPa, f = 10 kHz, Duty: 50% f U0 = -3 kV, f = 10 kHz, Pinput=2.75 W
100
0 0.4
50
Voltage (kV)
Current (A)
Charge (nC)
1 0.0
0
2 0.4
50
3 0.8 CDBD=7.8 pF
100
9.5 10.0 10.5 60.0 60.5 61.0 3 2 1 0 1
Time (ms) Voltage (kV)
Xe/I2=13.3/0.04 kPa, f = 60 kHz, Duty: 50%
g h U0 = 3.07 kV, f = 60 kHz, Pinput=17.52 W
100
3 0.4
50
Voltage (kV)
Current (A)
Charge (nC)
2 0.0
0
1 0.4
50
0 0.8
5 10 15 100
0 1 2 3 4
Time (ms) Voltage (kV)
Fig. 20.2 Measured voltage, current waveforms (a, c, e, g) and corresponding Volt-Coulomb
Lissajous figures (b, d, f, h) for a mixture Xe/I2 = 13.3/0.04 kPa under the different excitation modes
used in the experiments. Displacement current and switching noise are already subtracted for the
current waveform under pulsed excitation at f = 60 kHz (g)
20 Xenon Iodide Exciplex Lamp as an Efficient Source 271
Irradiance, mW/cm2
2
0 180 0
Lamp
2
4 210 330
6
240 300
8
270
almost the entire voltage cycle excluding short intervals in the vicinity of the peak
of the applied voltage (Fig. 20.2b).
The slope obtained for pulsed bipolar operation PS1 with Dtrise = 0.9 ms and
Dtfall = 0.6 ms is about 14 pF (Fig. 20.2d). This means that the discharge gap is already
slightly conductive during the ignition phase before the breakdown. When using
PS2, providing square pulses with short rising and falling times of ~50 ns, the UQ
Lissajous figure is a parallelogram with two vertical sections for On periods
(Fig. 20.2f). This is similar to the observed one for a plasma a display cell with
square wave voltage (f = 100 kHz) [14]. The slope dQ/dU = 7.8 pF coincides with the
calculated lamp capacitance. For unipolar pulsed operation with PS3 (Dtrise = 160 ns,
Dtfall = 60 ns), the dQ/dU for Off periods remains close to 8 pF, but at higher fre-
quency a breakdown occurs at lower voltage and the right On section in Fig. 20.2h
changes position from vertical to sloping.
Figure 20.3 shows the angular irradiance distribution of the DBD XeI* excilamp
measured at 1 m distance between the centre of the lamp and the sensor head. The
angular dependence shows that the total flux for the improved electrode system is
89% with respect to the isotropic light source which has Pmeas(90). The obtained
value also differs from a linear cylindrical light source (p/4 78.5%). We would like
to note an interesting feature from Fig. 20.3. The radiation from the ends of the
DBD XeI* excilamp does not fall to neglected values but has a significant effect into
the total radiant flux. Taking into account the lamp dimensions and emitting
272 M. Guivan et al.
10
Xe/I2 = 13.3/0.04 kPa
f = 60 kHz
8
XeI*(B - X)
)
3/2
o
I*( P5/2 - P
2
Intensity (a.u.)
) 1/2
6 o
I*( P3/2 - P
2
4
4
1/2
XeI*(B - A)
o
XeI*(C - A)
2
2
4
0
200 250 300 350
Wavelength (nm)
Fig. 20.4 VUV-UV emission spectrum of the excilamp with a Xe/I2 = 13.3/0.04 kPa mixture.
Bipolar pulses, Upp = 4.2 kV
surfaces (side Sside = 26.8 cm2 and end Send = 6 cm2), one can calculate that 1 cm2 area
provides, at L = 1 m, irradiance perpendicularly to the lamp axis Pmeas(90) = 0.28 mW/
cm2, while in the direction of the lamp axis Pmeas(0) = 0.5 mW/cm2. This should be
considered in the design of the reflecting fittings for the DBD-driven XeI*
excilamps.
In the experimental reports to date, the emission spectra of DBD-driven XeI* excil-
amps are presented mainly for wavelengths l > 200 nm. Recently Carman et al. [6],
based on computer modeling, predicted an efficient formation and intense radiation
of Xe2* excimer in the VUV range (l = 172 nm) from a Xe/I2 mixture. The overall
intrinsic conversion efficiency from electrical energy to UV output from the plasma
was calculated at 9.6% for 253 nm and 19.4% for 172 nm. We wanted to check this
prediction and investigate the VUV emission from a Xe/I2 mixture experimentally.
Figure 20.4 shows the spectra of the excilamp in the VUV and UV range operat-
ing with the optimized working mixture Xe/I2 = 13.3/0.04 kPa under pulsed excita-
tion (PS1). Measurements reveal the strong XeI*(BX) exciplex radiation at
lmax = 253 nm. Weaker emissions of XeI*(BA) (lmax = 320 nm), XeI*(CA)
(lmax = 265 nm), and I2*(DA) (lmax = 342 nm) were also observed. Atomic iodine
20 Xenon Iodide Exciplex Lamp as an Efficient Source 273
is represented in the spectrum by the emission lines in the range 178188 nm (178.3,
179.9, 183.0, 184.4, 187.6 nm) and at l = 206.2 nm (5p46s 5p5 transitions). It is
interesting that the observed 4P3/22P1/2 (184.4 nm) line intensity is negligible com-
pared to the most intensive 4P5/22P3/2 (183.0 nm) line contrary to the VUV spec-
trum of an AC excited DBD excilamp operated with a Kr/I2 = 40/1.33 kPa mixture,
where both lines were registered with similar intensity [12]. It also differs from
the emission spectrum of a DC glow discharge with a Xe/I2 mixture, where the
4
P5/22P3/2 (183.0 nm) line intensity counts only for 2% of the 2P3/22P1/2 intensity
at 206.2 nm [15].
We did not observe Xe2* excimer radiation from the XeI* excilamp (Fig. 20.4),
while a DBD on pure xenon at the same pressure and discharge conditions produced
intense broadband Xe2* emissions in the VUV region with a cut-off at l ~ 173 nm
due to the low transmittance of the quartz bulb. The XeI* and I2* emissions under
the present conditions do not fall to the background level but form a continuum in
the range 190360 nm. This might be important for practical applications of the
XeI* excilamp. Similar features have been reported before [16]. Recently, I2* emis-
sions produced by the DBD-driven excilamps were studied in detail [17, 18].
For all the excitation modes, the UV radiation power rises almost linearly with
increasing the input electrical power under the present discharge conditions
(Fig. 20.5a). Figure 20.5b shows the scaling of the excilamp efficiency with the
input power. The maximum efficiency obtained using pulsed unipolar excitation
with short pulse edges (PS2, PS3) is significantly (1.73.7 times) greater than that
obtained using AC excitation. In order to get the highest efficiency possible, the
voltage rising and falling times should be as short as possible. The operating fre-
quency affects the discharge appearance, and, as a result, the efficiency of the excil-
amp. The average radiation power of 10.3 mW/cm2 at the lamp surface was achieved
at f = 80 kHz.
20.4 Discussion
The presented results show that the enhanced performance of the XeI* excilamp is
derived from a discharge which is more diffuse than that achieved at the same Xe/I2
mixture pressures using AC excitation. The spatial distribution, UV power and effi-
ciency of the DBD-driven XeI* excilamp can be improved significantly by utilizing
pulsed excitation. Moreover, even for pulsed excitation the conversion efficiency is
very sensitive to the duration of voltage rising and falling times or, in other words,
274 M. Guivan et al.
UV Power (mW/cm2)
excitation modes: AC, 8
f = 60 kHz (1), pulsed bipolar
PS1, f = 60 kHz, 50% duty 6
(2), pulsed unipolar PS2, 2
f = 10 kHz, 50% duty (3), 4 3 4
and pulsed unipolar PS3, 1
f = 60 kHz, 50% duty (4). 2
Xe/I2 = 13.3/0.04 kPa mixture a
0
Efficiency (%) 12
8 3
4
4 2
1 b
0
0 4 8 12 16 20
Input power (W)
to the dU/dt. Small increases of Dtrise by about 100 ns (for pulsed excitation) cause
changes in the discharge pattern and UV output. Moreover, for pulsed operation the
current amplitude is determined by the pulse edge steepness. The application of
fast-rising square voltage pulses to the discharge gap allows the discharge to occur
more homogeneously throughout the entire active length of the excilamp.
Comparison of the electrical characteristics (UI and UQ) depicted in Fig. 20.2
and excilamp efficiency (Fig. 20.5) show that conditions for high efficiency are
obtained for the excitation modes corresponding to the near vertical lines for On
periods in the UQ Lissajous figures.
The mechanism of efficiency enhancement compared to that presented in [4, 6]
has been described in [19]. Excellent coincidence in the temporal behavior of the
XeI*(BX) emission and the Xe(1 s5) absorption has proved that in DBD under the
present conditions a harpoon reaction is the dominant source of XeI*(B) population.
We have diminished the effect of the unwanted formation of Xe2*(3S) and Xe2I* by
operating in lower xenon pressures p and with a larger discharge gap d in order to
sustain the optimal value of the pdparameter. Neither Xe2* nor Xe2I* emission was
registered in the spectrum of the developed XeI* excilamp with the optimized work-
ing mixture (Fig. 20.4). This is why we used a gap 2.12.6 times larger than in [6].
We think that it is one of the main sources of efficiency improvement compared to
the values presented in [6]. Another reason is the use of pulsed excitation with shorter
20 Xenon Iodide Exciplex Lamp as an Efficient Source 275
104
rising and falling times. We can assume that under these excitation modes (PS2 and
PS3) the most appropriate discharge conditions (mean electron energy, electron tem-
perature, electron energy distribution function (EEDF)) are created for the conver-
sion of ground state Xe atoms into low-level Xe*(6 s) states and to prevent energy
losses associated with stepwise excitation, ionization and ion heating [20].
The developed XeI* DBD excilamp can be successfully used for practical appli-
cations, such as UV cleaning, surface treatment and UV sterilization. The glass
plates were chosen as the test objects for the UV surface cleaning. It can be seen
from the Fig. 20.6 that after 1 min irradiation the water drop on the glass surface has
a contact angle about three times less in comparison with the untreated surface.
It means, the UV radiation from the XeI* lamp destroyed the oil film on the surface
and can be used for the UV cleaning.
Figure 20.7 shows the changes in the normalized number of CFU in the water
flow reactor as a function of the cumulative UV dose of XeI* excilamp, emitted dur-
ing the UV treatment. One experiment was carried out when the volume of irradiated
water was covered with a plate preventing the penetration of foreign microflora from
outside, and another one without a cover. Almost the same CFU reduction was
276 M. Guivan et al.
20.5 Summary
~50 ns was 9%, which is about 2.7 times greater than the maximum efficiency under
AC excitation (3.3%). In order to reach the highest efficiency, the voltage rising and
falling times should be as short as possible, and plasma breakdown has to occur
close to the peak of the voltage pulse.
The sterilization action of the DBD-driven XeI* excilamp was tested on the inac-
tivation of B. subtilis spores. A reduction by more than 4 orders of magnitude of
CFU in B. subtilis spores was achieved in a water flow reactor and the D-value was
about 0.4 J/cm2. An additional effect of the I* radiation at 206 nm and in the VUV
range (178188 nm) was confirmed. This research demonstrates that the DBD-
driven XeI* excilamp can be used for the UV cleaning and inactivation of microor-
ganisms in movable systems (drinking water treatment or food package
sterilization).
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Part IV
Plasma Tissue Treatment
and Wound Healing
Chapter 21
Antisepsis of the Skin by Treatment with
Tissue-Tolerable Plasma (TTP): Risk
Assessment and Perspectives
Abstract The application of tissue tolerable plasma (TTP) is well suited for
disinfection of living tissue. In particular, when treating chronic wounds, it has
several advantages in comparison to the classical application of antiseptics, which
do not penetrate sufficiently into the tissue or inhibit wound regeneration. The mode
of action of the plasma is mainly based on synergetic effects between temperature
increase and the formation of free radicals, which destroy the bacteria and fungi.
In the present paper a risk assessment of TTP in dermatology is given. The inves-
tigations have been carried out with an atmospheric pressure plasma-jet working
with Argon as a discharge medium. It was found that during the plasma treatment of
tissue, the antioxidative potential is reduced only in the upper part of the stratum
corneum, but not in deeper cell layers. Selecting the optimum parameters of the
plasma formation, the UV exposure of the skin is less than in the case of UV irradia-
tion of the sun on a summer day at noon.
If the duration of the plasma treatment of the skin is in the optimal range for
wound healing, no thermal damage has to be expected.
Additionally, it could be demonstrated that plasma is able to reach the follicular
reservoir for antisepsis where germs are located.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 281
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_21, Springer Science+Business Media B.V. 2012
282 J. Lademann et al.
21.1 Introduction
1. As the result of a gaseous discharge, radicals are produced on the skin surface.
The human skin is continuously exposed to free radicals, which are produced
by environmental factors such as UV sun irradiation [6]. The human skin has
developed a protection system against the destructive action of these highly
reactive molecules in form of the antioxidative network. Therefore, the influ-
ence of the plasma on this antioxidative network of the skin was
investigated.
2. During the plasma formation, also UV-irradiation is produced [7]. The emission
spectrum of the plasma on the skin surface and in different depths of model
tissue (pig ear skin) was investigated depending on different physical parameters
influencing the formation of the plasma, such as gas pressure, discharge
voltage.
3. In a series of experiments, the influence of the duration of the plasma treatment
on the temperature of the skin surface was analyzed relating to different physical
properties influencing the plasma parameters (e.g., gas pressure, discharge
voltage).
All investigations excluding the analysis of the antioxidants were performed on pig
ear skin, which is a suitable model for human tissue [12]. Fresh pig ears had been
obtained from a nearby butcher. Before being used in the experiments, the pig ears
were washed with cold water and dried with paper towels. Sliced skin samples, with
a thickness of 200 mm and a size of 3 3 cm, were obtained from the pig ears by
using the Dermatom GA 148 (AESCULAP AG and Co. KG, Tuttlingen,
Germany).
Approval for the experiments had been obtained from the Veterinary Board,
District of Berlin-Koepenick.
The analysis of the influence of the plasma treatment of the antioxidants was
carried out in vivo on six volunteers, because it was demonstrated previously that
the radical formation in necrotic tissue is strongly reduced in comparison to the
in vivo situation. The volunteers were aged between 28 and 47 years. Approval for
the study had been obtained from the Ethics Committee of the Charit -
Universittsmedizin Berlin, Germany.
21.2.2 Plasma-Jet
In the present study, the plasma-jet (kinpen 09) was utilized, which had been
developed at the Institute of Plasma Physics, Greifswald, Germany and manufac-
tured by Neoplas GmbH [13, 14]. The plasma-jet has been described in detail previ-
ously [4, 14]. Argon gas was used as a discharge medium in the plasma-jet. The gas
flow was 4 standard liters per minute (slm). The applied voltage was 170 V. The top
of the plasma stream acts in the mode of a brush during the treatment of the skin.
The plasma stream had a length of 17 mm, whereas the plasma-tissue interaction
zone was approximately 1 mm in diameter, depending on the nozzle-to-tissue dis-
tance. In Fig. 21.1, the nozzle of the plasma-jet and the plasma stream are shown.
The device kinpen 09 has CE-Certification for fulfilling the standard of
electrical safety in humans.
To evaluate the plasma emission in the tissue, the spectra of the plasma beam were
analyzed behind layers of corneocytes removed by tape stripping and behind sliced
skin. Tape stripping is a well suited method for removal of single cell layers of the
stratum corneum of the skin [15]. Adhesive films (Tesa Film No. 5529, Beiersdorf
284 J. Lademann et al.
AG, Hamburg, Germany) were applied and successively removed from the same
skin area. By repeating this procedure, the stratum corneum could be removed
sequentially, cell layer by cell layer.
The spectrum of the plasma emission was analyzed in the spectral range between
200 and 600 nm, using a fiber-based spectrometer EPP2000 (SI Scientific Instruments
GmbH, Gliching, Germany). The optical quartz fiber (Laser- und Medizin-
Technologie GmbH, Berlin, Germany) contains a scattering body at the fiber tip,
which collects the surrounding light and transfers it to the fiber.
The temperature of the plasma stream on the skin surface was determined using a
digital thermometer GTH 1200A (Greisinger electronic GmbH, Regenstauf,
Germany). The measuring spot was approx. 1 mm in diameter.
skin treatment with octenidine and TTP. The investigations were carried out on skin
areas, which remained untreated, on skin areas treated with the antiseptic octenidine
and on TTP treated skin. Immediately after treatment, the procedure for the deter-
mination of the amount of bacteria on the skin surface started in accordance with the
Williamsons protocol [16].
For an efficient disinfection it is necessary that plasma is able to reach the follicular
reservoir to destroy the bacteria and fungi, which are localized in this area. The
investigations were performed on porcine skin by using a solution containing a
chlorophyll dye. The fluorescent properties of the dye changed during the plasma
tissue interaction, as described by Lademann [10].
Histological sections were obtained from biopsies removed from the non-treated
and plasma-treated tissue samples. The fluorescence signal of the dye in the hair
follicles was analyzed in both cases using laser scanning microscopy.
The structure of the skin surface of the tissue samples, before and after plasma treat-
ment, was analyzed by laser scanning microscopy (Stratum, Optilas Ltd.,
Melbourne, Australia). The aim of these investigations was to investigate whether
there is a thermal damage to the skin surface after plasma treatment. The laser scan-
ning microscope consists of a base station containing the excitation laser (argon
laser, l = 488 nm) and the spectrometer in the control unit [17]. The base station is
connected by optical fibers to the hand piece, where the optical imaging system and
the focus control unit are positioned. The maximal penetration depth of the radia-
tion into the tissue was approximately 150 mm, which implies that the skin could be
analyzed up to the upper papilla structure [17]. A detailed description of the laser
scanning microscope is given by Kandarova et al. [18].
The radical formation during plasma treatment was analyzed indirectly by deter-
mining the concentration of the carotenoids in the skin.
These investigations were undertaken in vivo on the forearms of healthy human
volunteers, because the radical formation in necrotic tissue is strongly reduced on
account of its low oxygen concentration. The radicals formed in the tissue react with
286 J. Lademann et al.
the antioxidants, including the carotenoids. These highly reactive molecules are
neutralized by the antioxidants, before they can damage cells or cell compartments.
If the concentration of the free radicals exceeds a critical value, the antioxidants are
destroyed and consequently their concentration is reduced. By means of this proce-
dure, the carotenoids can be used as marker substances for radical formation in the
skin.
The carotenoid concentration was determined in different depths of the stratum
corneum by means of the Raman laser scanning microscope (River Diagnostics,
Rotterdam, Netherlands)
Using the optimal operation parameters of the plasma-jet, the risk aspects were
evaluated regarding UV-radiation, temperature effects and radical formation.
The emission spectrum of the plasma consists of one intensive line at 310 nm and
several smaller bands between 325 nm and 450 nm (Fig. 21.2). The emission signal
at 310 nm results from the OH radicals. Using the described plasma-jet system with
Argon as a charge medium, no emission bands at wavelengths lower than 300 nm
could be detected. The UV light at 310 nm is efficiently absorbed by the stratum
corneum of the skin. A single corneocyte layer removed by tape stripping reduces
the transmittance to 25% of the plasma radiation at 310 nm. The stratum corneum
of the human skin consists of 1525 cell layers, depending on the body site. This
means that almost no UV-radiation reaches the living cells of the skin. This was also
confirmed by transmission measurements of sliced skin [19].
The same spectroscopic arrangement, which was applied in the plasma
experiment, was used to measure the intensity of the sun radiation, on a sunny day
in March at noon. Comparing the sun radiation and the plasma emission, it was
found that the radiation dose produced by the plasma-jet at 310 nm was one order
of magnitude below the minimal erythema dose, necessary for the formation of skin
damage detectable as sunburn.
The temperature determined during the plasma treatment on the skin surface was
between 35C and 45C depending on the moving velocity. Changes in the cellular
structure of the stratum corneum caused by thermal effects could be detected only
at low moving velocities in the first two cell layers of corneocytes. This demon-
strates that the thermal action of the plasma is highly superficial and is limited to the
stratum corneum without affecting the living tissue.
Analyzing the carotenoid concentration in different depths of the stratum
corneum, before and after plasma treatment, it was found that the carotenoids were
partly destroyed only in the upper cell layers of the stratum corneum. In the
corneocyte layers close to the living epidermis no changes in the carotenoid level
could be observed [20]. This means that the radical formation is a superficial effect.
21 Antisepsis of the Skin by Treatment with Tissue-Tolerable Plasma (TTP) 287
200
180
160
Intensity [arb. units]
140
120
100
80
60
40
20
0
200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
Wavelength [nm]
The radicals are produced in high concentrations on the skin surface where the bac-
teria and fungi are located. In Fig. 21.3, the typical change of the carotenoids con-
centration in the stratum corneum of one volunteer prior to and after plasma
treatment is demonstrated. Additionally, as shown by histological investigations the
plasma penetrates effectively into the hair follicles [21], where radical formation
processes can be expected as discussed below.
140
before plasma treatment
after plasma treatment
120
Carotenoid concentration [m]
100
80
60
40
20
0
0 2 4 6 8 10 12 14
Depth of the SC [m]
Fig. 21.3 Carotenoid concentration in the stratum corneum prior to and after plasma treatment
Fig. 21.4 Determination of the bacterial colonies on agar plates before (a) and after (b) plasma
treatment
can be explained by the small size of the plasma beam of 2 mm. On account of this
small diameter, a thorough and homogeneous disinfection of the complete skin sur-
face seems difficult. It cannot be excluded that small residues on the skin surface
remained unaffected by the plasma stream during treatment. Consequently, in all
probability, bacteria may have survived in these skin areas leading to an increased
number of bacterial colonies.
21 Antisepsis of the Skin by Treatment with Tissue-Tolerable Plasma (TTP) 289
120
100
Efficancy of disinfection [%]
80
60
40
20
0
Octenidine Plasma jet
Fig. 21.5 Efficacy of disinfection after in vivo treatment of the skin with octenidine and plasma
21.4 Conclusions
The treatment of the skin with tissue tolerable plasma is a highly efficient method
for skin disinfection. The diameter of the plasma-tissue interaction zone must be
selected in such a manner that the tissue surface is homogeneous and completely
treated. In comparison to classical application of antiseptics, the TTP has the
advantage of disinfecting also the hair follicles, which are a reservoir for bacteria
and fungi.
The temperature increase on the skin surface during the plasma treatment is
moderate. The thermal damage was observed or was related only to the upper cell
layers of the stratum corneum. The UV exposure of the skin by the investigated
plasma treatment was one magnitude below the erythema level. Also, the radical
formation during the plasma treatment is located only on the skin surface, sufficient
enough to destroy the bacteria and fungi. However, the radicals do not damage the
living epidermis. Consequently, TTP has a high perspective in medicine for disin-
fection and wound healing. No risk potentials are to be expected for the patient
when the system investigated in this study is applied in vivo.
290 J. Lademann et al.
Acknowledgments This work was realized within the framework of the multi-disciplinary
research cooperation of Campus PlasmaMed, particularly within the project PlasmaWound.
The authors acknowledge that this work was supported by a grant funded by the German Ministry
of Education and Research (BMBF, Grant No, 13 N9779).
References
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27(7):611621
2. Sekkat N, Kalia YN, Guy RH (2002) Biophysical study of porcine ear skin in vitro and its
comparison to human skin in vivo. J Pharm Sci 91(11):23762381
3. von Woedtke T, Kramer A, Weltmann KD (2008) Plasma sterilization: what are the conditions
to meet this claim? Plasma Process Polym 5(6):534539
4. Weltmann KD, Kindel E, Brandenburg R, Meyer C, Bussiahn R, Wilke C, von Woedtke T
(2009) Atmospheric pressure plasma jet for medical therapy: plasma parameters and risk esti-
mation. Contrib Plasma Phys 49(9):631640
5. Weltmann KD, Kindel E, von Woedtke T, Hahnel M, Stieber M, Brandenburg R (2010)
Atmospheric-pressure plasma sources: prospective tools for plasma medicine. Pure Appl
Chem 82(6):12231237
6. Zastrow L, Groth N, Klein F, Kockott D, Lademann J, Renneberg R, Ferrero L (2009) The
missing linklight-induced (2801,600 nm) free radical formation in human skin. Skin
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7. Lademann J, Richter H, Alborova A, Humme D, Patzelt A, Kramer A, Weltmann KD,
Hartmann B, Ottomann C, Fluhr JW, Hinz P, Hubner G, Lademann O (2009) Risk assessment
of the application of a plasma jet in dermatology. J Biomed Opt 14(5):054025
8. Lademann O, Richter H, Patzelt A, Alborova A, Humme D, Weltmann KD, Hartmann B,
Hinz P, Kramer A, Koch S (2010) Application of a plasma-jet for skin antisepsis: analysis of
the thermal action of the plasma by laser scanning microscopy. Laser Phys Lett
7(6):458462
9. Hammann A, Huebner NO, Bender C, Ekkernkamp A, Hartmann B, Hinz P, Kindel E, Koban I,
Koch S, Kohlmann T, Lademann J, Matthes R, Muller G, Titze R, Weltmann KD, Kramer A
(2010) Antiseptic efficacy and tolerance of tissue-tolerable plasma compared with two wound
antiseptics on artificially bacterially contaminated eyes from commercially slaughtered pigs.
Skin Pharmacol Physiol 23(6):328332
10. Lademann O (2011) Antisepsis of the follicular reservoir by treatment with tissue-tolerable
plasma (TTP). Laser Phys Lett 1-5. doi:10.1002/lapl.201010123
11. Lange-Asschenfeldt B, Alborova A, Kruger-Corcoran D, Patzelt A, Richter H, Sterry W,
Kramer A, Stockfleth E, Lademann J (2009) Effects of a topically applied wound ointment on
epidermal wound healing studied by in vivo fluorescence laser scanning microscopy analysis.
J Biomed Opt 14(5):054001
12. Meyer W, Schwarz R, Neurand K (1978) The skin of domestic mammals as a model for the
human skin, with special reference to the domestic pig. Curr Probl Dermatol 7:3952
13. Foest R, Kindel E, Ohl A, Stieber M, Weltmann KD (2005) Non-thermal atmospheric pressure
discharges for surface modification. Plasma Phys Control Fusion 47:B525B536
14. Weltmann KD, Brandenburg R, von Woedtke T, Ehlbeck J, Foest R, Stieber M, Kindel E
(2008) Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pres-
sure plasma jets (APPJs). J Phys D Appl Phys 41(19):194008
15. Lademann J, Jacobi U, Surber C, Weigmann HJ, Fluhr JW (2009) The tape stripping procedure
evaluation of some critical parameters. Eur J Pharm Biopharm 72(2):317323
16. Williamson PT, Roth JF, Haddingham T, Watts A (2000) Expression and purification of recom-
binant neurotensin in Escherichia coli. Protein Exp Purif 19(2):271275
21 Antisepsis of the Skin by Treatment with Tissue-Tolerable Plasma (TTP) 291
In recent years, the range of atmospheric pressure applications for medical and
biological purposes is growing fast, and a new field of Plasma Medicine was formed
[15]. Various types of plasmas, both thermal and non-thermal, are now widely
studied for the purposes of blood coagulation [2, 6, 7], sterilization of living tissues
[1, 2, 6], treatment of various wounds and burns [2, 6, 8], gastrointestinal diseases [9],
and even cancer [10]. This opens up new horizons in both medical and physical
D. Dobrynin (*)
Electrical and Computer Engineering Department, Drexel University,
3141 Chestnut Street, Philadelphia, PA 19104, USA
e-mail: dvdobrynin34@gmail.com
G. Fridman G. Friedman A. Fridman
Drexel Plasma Institute, Drexel University, 3141 Chestnut Street,
Philadelphia, PA 19104, USA
e-mail: fridman@drexel.edu
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 293
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_22, Springer Science+Business Media B.V. 2012
294 D. Dobrynin et al.
Fig. 22.1 General schematic of the Pin-to-Hole spark Discharge (PHD) plasma system and a
photograph of the discharge in operation
using an LS55 (Perkin Elmer) fluorescent spectrometer equipped with well plate
reader accessory.
Agarose gel has been traditionally used to mimic biological substrates like
tissues, skin, cell layers, etc. Although it does not represent real tissue we show
that one is able to alter the gels buffering ability, density and fluidity to closely
resemble tissue. Agarose gels of 1.5% wt are prepared using standard procedure
with pure agar powder (Fisher) in either distilled water or phosphate buffered
saline (PBS, Fisher). Measurements of H2O2 penetration into agarose gels and
tissues were done using Amplex UltraRed reagent (Invitrogen, ex/em:
530/590 nm) fluorescent dye. 75 mL PBS containing 100 mM Amplex UltraRed
with 200 U/mL horseradish peroxidase (MP Biomedicals) were placed in between
1 mm thick 4 4 cm agar slices and incubated for about 15 min before the treat-
ment in order to provide presence of the dye in the agar volume. In order to
measure the H2O2 in tissue, the dye was injected using a syringe into a 1 cm thick
4 4 cm skinless chicken breast tissue samples at various points to the depth of
up to 1 cm. Treated samples were sliced in a vertical direction with thickness of
1 mm, and fluorescence was measured using an LS55 (Perkin Elmer) fluorescent
spectrometer equipped with XY reader accessory (Fig. 22.2). To obtain calibra-
tion curves for hydrogen peroxide in the plasma treated samples a standard sta-
bilized 3% H2O2 (Fisher) water solution properly diluted to obtain various
concentrations was used.
In Fig. 22.3 we report the results of plasma production of H2O2 (with and without
presence of superoxide dismutase), and NO delivery into PBS solution is shown in
Fig. 22.4. In order to ensure that the H2O2 specific dye is not altered by UV radiation
produced by plasma, first measurement was done through a quartz glass. As shown
296 D. Dobrynin et al.
Fig. 22.2 Chicken breast after plasma treatment with H2O2 fluorescent dye: photograph and fluo-
rescent images from the top and side of the sample (intensity is in arbitrary units, position of the
sample was different during measurements)
Fig. 22.3 Production of hydrogen peroxide and superoxide in PBS (100 ml) by microsecond spark
discharge
22 Cold Microsecond Spark Discharge Plasma Production of Active Species 297
Fig. 22.4 Delivery of nitric oxide as measured by fluorescent dye DAF-2 in PBS (100 ml) by
microsecond spark discharge
in Fig. 22.3, the microsecond spark discharge produces a relatively stable amount
of about 40 mM of hydrogen peroxide in the solution. Addition of superoxide
dismutase (SOD) which catalyzes superoxide dismutation reaction
2O2 + 2 H +
SOD
H 2O2 + O2 , allows indirect measurement of O2 (Fig. 22.3). In
Fig. 22.4 we show that together with ROS we measure a significant amount of NO
produced by plasma in the PBS (compared to 8,000 ppm NO balanced with N2 from
a tank, Air Products) Simultaneous production of both ROS and RNS, and specifi-
cally H2O2 and NO may result in a number of biologically important effects. For
example, it has been shown, that presence of both NO and H2O2 may induce
apoptosis in cancerous cells [13], inactivate bacteria [14], and form biologically
important singlet oxygen [15].
The results of H2O2 measurements in chicken breast tissue are shown in
Fig. 22.5: with longer treatment time depth of penetration as well as concentra-
tion of hydrogen peroxide increases. In general, several millimoles per liter of
H2O2 are produced in tissue after plasma treatment, while it diffuses 1.53.5 mm
deep. The measurement results for H2O2 produced by plasma treatment in agar
gels are shown on Fig. 22.5. Hydrogen peroxide concentration on the agar gel
surface was 1.9 mM after 1 min of plasma treatment. The difference of the H2O2
concentration profile may be related to actual structural differences between aga-
rose gel and tissue, in which macroscopic irregularities (e.g. fibers, pores, etc.)
are present.
298 D. Dobrynin et al.
7
tissue 30s
tissue 60s
6 tissue 120s
120s agar 30s
5 agar 60s
agar 120s
[H2O2], mM
60s
3
2
30s
0
0 1 2 3 4 5 6 7
Depth, mm
Fig. 22.5 Hydrogen peroxide penetration depth in tissue and agarose gel
22.3 Conclusion
The results show that PHD discharge effectively produces both ROS and RNS
species in the media, and active species may be delivered into the tissues to the
depths of several mm, therefore providing not only surface effects (inactivation of
pathogens, first of all), but also therapeutic effects inside of treated tissues. The
agarose tissue model shows that plasma effects may be transferred several millime-
ters deep inside a tissue, as measured in an ex vivo chicken tissue model. We have
detected a penetration behavior of a simplest active component, hydrogen peroxide,
but other species may be detected and measured using other fluorescent dyes readily
available. In addition, we showed that a simple agar gel model may express similar
physicochemical properties as a real tissue, resulting in comparable penetration
effects of active species.
References
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Huang (2009) Blood clotting by low-temperature air plasma. IEEE Trans Plasma Sci
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7. Kalghatgi SU, Fridman G, Cooper M, Nagaraj G, Peddinghaus M, Balasubramanian M,
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Chapter 23
Surface Dielectric Barrier Discharge
Jet for Skin Disinfection
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 301
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_23, Springer Science+Business Media B.V. 2012
302 Y. Creyghton et al.
The plasma jet source has been constructed using an alumina ceramic tube with an
inner temperature conditioned high voltage conductor. Two parallel flattened sur-
faces of the initially circular tube form two series of a thin electrical gas discharge
spaces with the adjacent exterior electrode as shown in Fig. 23.1. The exterior elec-
trodes have protruding ribs touching the ceramic and serving as starting points for a
large number density of surface dielectric barrier discharges (SDBD) on the ceramic
surface. The surface discharges are created in volume spaces of 5 5 S mm3 where
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection 303
Fig. 23.1 Cross sectional view of the SDBD electrode structure. The gas flow path is oriented in
perpendicular direction through narrow discharge spaces (5.0 0.5 mm2)
different metal electrode structures allow a lit width S ranging from 50 to 500 mm.
The active length of the electrode is 300 mm. The source offers various options:
It can be operated under a wide variety of electrical conditions (AC + DC, repeti-
tively pulsed), gases (N2-O2, noble gases) and gas flow conditions. SDBD plasma
is stable as function of time and homogeneously distributed in space.
The elongated electrode structure enables fast treatment of a large surface area
using a single unidirectional displacement.
The high speed at moderate gas flow of the thin sheet of plasma activated gas
allows treatment at relatively large distances, up to 50 mm.
Temperature conditioning of the ceramic core allows high power and improved
control of the ratio of reactive nitrogen to reactive oxygen species [RNS]/[ROS].
The high voltage electrode is effectively screened from the earthed external elec-
trodes which can be safely touched under plasma operation.
Since the SDBD plasma jet electrode structure provides a gas flow over its entire
length of 300 mm, it is necessary to remove the surplus of gas from the treating
volume. For this purpose a gas withdrawal system has been provided. This system
consists of two gas sucking elements placed at both sides of the electrode configura-
tion, as shown in Fig. 23.2. Alternatively the sample holder plate is replaced by a
gas permeable screen and gas removal elements are placed below this screen.
Substrates are treated by the plasma jet after placing them on a sample holder
which is positioned on a movable sample holder support plate. The time period of
plasma treatment is controlled by means of the moving speed of the sample. Before
or after plasma treatment the sample can be humidified by passing the sample
through an aerosol spray cabin as shown in Fig. 23.2.
The liquid spray unit (Spray Systems Inc.) provides a 300 50 mm2 wide spray
pattern. The liquid and air pressure conditions of the flat spray type nozzle have
been optimized for homogeneous spraying of <100 mm size mist droplets. The minimum
sprayed liquid density is 0.05 mg/cm2. Liquid density fluctuations over the treating
304 Y. Creyghton et al.
Fig. 23.2 Experimental configuration showing the relative positions of the SDBD electrode struc-
ture, gas removal elements, the spray cabin and the movable sample holder plate. Parameters D and
d design the vertical and lateral distance of a treating point on a substrate with respect to the centre
of the source nozzle
area are determined at less than 23%. A special shape of the spray cabin allows for
the use of the central most homogeneous part of the spray cone by removal of spray
cabin wall deposited liquid.
The line shaped plasma jet nozzle with 300 mm length (Fig. 23.3) is positioned
at adjustable distance D from the surface to be treated. For microbial inactivation
tests, for instance five stainless steel sample holders (25 mm in diameter) can be
disposed in a row receiving similar treatment from the linear source. In addition, as
shown in Fig. 23.2, a temperature probe and a gas sample point for ozone and NOx
analysis have been included. The BMT930 ozone monitor has been calibrated up to
concentration levels of 20 ppm. It has a 15 s sample measurement time which is
acceptable for obtaining an indication of ozone at the location of the sample when
moving the sample holder plate at a low speed of 1 mm/s. The temperature of the
liquid inside the core of the high voltage electrode is measured with a Neoptix opti-
cal fiber system.
A Pillar CS6030 power supply has been used in combination with a HT3 trans-
former from ITW Surface Treatment to supply voltage and current to the plasma.
The required amplitude of the voltage is 6 kV. For the given capacitance of dis-
charge configuration (~200 pF) the power supply was controllable from 50 to
600 W (maximum frequency was 25 kHz). Initial tests have been performed using
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection 305
Table 23.1 Temperatures (C) as function of vertical distance (D) and horizontal distance (d)
from the plasma jet nozzle
500 W
40 L/min d = 0 mm (C) d = 10 mm (C) d = 30 mm (C) d = 100 mm (C)
D = 0 mm 44.5
D = 20 mm 33.2 23 23 22.8
D = 40 mm 22.3 21 21 20.9
The substrate plate velocity is 1 mm/s
In order to design a gas removal system for the application of hand disinfection
in combination with the linear SDBD plasma jet source, the temperature and flow
fields have been calculated using the finite element software package Comsol. An
initial result of a flow field calculation is shown in Fig. 23.4, showing the flow pat-
tern through and around two fingers represented by cylindrical rods at 2 cm distance
of the source nozzle.
In the calculated flow pattern a vortex zone is observed with a gas recirculation
zone directly downstream the slit shaped nozzle. While the flow velocity in the two
parallel electrode slits of the plasma jet reaches initially 2 m/s, the two gas streams
leaving the slits (at 6 mm distance) confine to a single stream with a velocity in the
0.20.4 cm/s range. The flow velocity at the back side of fingers is far below 0.1 m/s
thus showing that movement of hands or various plasma jets are required to obtain
a sufficient treatment of the hand surface.
The test surfaces are glass slides (contaminated area = 20 35 mm2) or stainless steel
discs with a diameter of 20 or 25 mm. Prior to use, the slides or discs are cleaned by
placing them in 5% decontaminating solution and sonicated for 15 min. The discs
are rinsed with purified water and sterilized by autoclaving using moist heat (15 min,
121C).
Small agar plates are inoculated with bacteria or spores thereof and treated with
a various number of plasma treatment conditions. From a serial dilutions series,
agar plates are inoculated to a concentration of 105, 104, 103 or 102 cfu per plate. The
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection 307
Fig. 23.4 Calculated gas flow in between and around two fingers 6 mm apart from each other
and at 20 mm distance from the line shaped nozzle. The calculation assumes plane symmetry
across the plane at the left picture border. The numbers on the left and bottom are in units of meter.
The flow velocity (right hand side bar) is given in m/s. Applied flow condition: 40 L/min per tube
(20 L/min through each slit), 300 mm length. The whole bottom area is used for gas withdrawal
with a total flow rate twice the gas flow rate via the electrode
plates have been incubated and the number of surviving germs is counted. Agar
plates are incubated at 3537C for 2448 h. The data, compared to non treated
samples give an estimate of the bactericidal and sporicidal activity of the treatment.
The colony counts of the positive control are use as baseline to calculate the achieved
reduction. This procedure has applied for E. coli, S. aureus and spores of B. globigii
and B. subtilis.
E. coli and B. globigii have been treated in dry form on glass slides. First results as
shown in Fig. 23.5 indicate that ~20 s treatment (25 mm samples with 2 mm/s) at
10 mm distance from the source nozzle results in a significant reduction of colony
forming units from E. coli. A thin film of distilled water (~0.1 mg/cm2) causes a
dramatic further increase of the inactivation efficiency. Replacing water with a 5%
chloroxylenol solution (Dettol) is shown to cause a small but significant synergetic
effect. A one log decrease of B. globigii spores has been observed in this case as well.
Figures 23.6 and 23.7 show the influence of the percentage oxygen and the treat-
ment time respectively. With a decreasing amount of oxygen, inactivation decreases
for all types of microorganisms, however in pure nitrogen a single log inactivation
is maintained.
308 Y. Creyghton et al.
Fig. 23.5 Biocidal effects for E. coli treated by a 500 W plasma jet with 2 mm/s sample move-
ment. P, NP design plasma and no plasma treatment, S, NS design spray and no spray treatment.
The percentage oxygen concentration in nitrogen has been varied. The distance D from the source
nozzle is 10 mm. For each condition a minimum and maximum CFU count result are shown,
representing the statistic variation in CFU count results
Fig. 23.6 Microbiological inactivation as a function of O2% in N2-O2 gas mixture. The distance D
from the source nozzle is 15 mm. The log reduction values are average values of five samples
treated simultaneously by the plasma jet system
23 Surface Dielectric Barrier Discharge Jet for Skin Disinfection 309
Fig. 23.7 Microbiological inactivation as a function of substrate velocity. Other conditions are the
same as given with Fig. 23.6
This initial investigation of the SDBD plasma jet source shows that effective and fast
disinfection treatment of a large three dimensional object such as a human hand is
feasible at acceptable temperatures and in a reasonable short period of time (<2030 s).
310 Y. Creyghton et al.
One of the process parameters which need further optimization is the gas inflow rate
which can be strongly reduced by making the slit width between the ceramic tube and
the external electrodes of the source much smaller. The combination of mild liquid or
cream disinfectants with plasma dissociation which may cause the creation of addi-
tional reactive species is giving new perspectives. An important effort is still to be made
to control gas flow patterns in a treating device in order to minimize gas consumption,
gas removal and/or circulation without exposing the environment to unacceptable
levels of stable gaseous plasma products such as ozone and nitrogen oxides.
References
1. Nosenko T et al (2009) Designing plasmas for chronic wound disinfection. New J Phys 11.
http://iopscience.iop.org/1367-2630/11/11/115013
2. Kalghatgi S (2011) Effects of non-thermal plasma on mammalian cells. PLoS One 6:e16270
3. Bartosz G (2009) Reactive oxyfen species: destroyers or messagers? Biochem Pharmacol
77:13031315
4. Ross C et al (2006) Involvement of reactive oxygen species and reactive nitrogen species in the
wound response of Dasycladus vermicularis. Chem Biol 13:353364
5. Liebmann J et al (2011) Biological effects of nitric oxide generated by an atmospheric pressure
gas-plasma on human skin cells. Nitric Oxide 24:816
6. Lee HW et al (2011) Modelling of atmospheric pressure plasmas for biomedical applications. J
Phys D Appl Phys 44:127
7. Gadri RB et al (2000) Sterilization and plasma processing of room temperature surfaces with a
one atmosphere uniform glow discharge plasma. Surf Coat Technol 131:528542
8. Creyghton Y et al (2008) A surface dielectric barrier discharge plasma unit and a method of
generating a surface plasma. Patent application number WO2008082297
Chapter 24
Cold Atmospheric Plasma for Clinical
Purposes: Promising Results in Patients
and Future Applications
Georg Isbary
Abstract Infected chronic wounds are both socioeconomic and medical problem.
Cold atmospheric plasma (CAP) has already proven its efficacy in killing bacteria on
agar plates but also the first prospective randomized controlled trial in patients. As an
add-on therapy CAPs proved a highly significant decrease in bacterial load in 5 min
plasma-treated wounds (34%, p < 106, n = 291, 36 patients) in comparison with
wounds that received only standard wound care. This reduction is found in all kinds
of germs, even multiresistant ones. Two minutes of plasma treatment led to a signifi-
cant reduction in bacterial load as well (40%, p < 0.016, n = 70, 14 patients). The
treatment is very well tolerated and no side effects occurred until now (in total more
than 2,000 treatments in over 220 patients). The results of this study revealed the
potential of atmospheric argon plasma treatment as a new approach to kill bacteria in
terms of mutiresistancy. With the same CAP device other dermatologic diseases were
treated successfully, e.g. Hailey-Hailey disease. New plasma devices using surround-
ing ambient air have not only greater bactericidal but also virucidal properties. These
devices may herald a new era in public, personal, pet, and food hygiene, same as in
decontamination. Investigations of human compatibility are promising.
24.1 Introduction
G. Isbary (*)
Department of Dermatology, Allergology and Environmental Medicine,
Hospital Munich, Koelner Platz 1, 80804 Munich, Germany
e-mail: dr.isbary@googlemail.com
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 311
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_24, Springer Science+Business Media B.V. 2012
312 G. Isbary
antibiotics. However, bacteria stroke quickly back and became rapidly resistant towards
the new panaceas. Nowadays, in twenty-first century, it is daily routine at health care
facilities to verify resistance levels of bacteria before antibiotic treatment and to find
isolated patients due to infections with multiresistant germs in hospitals all over the
world. To treat bacterial infections has become a challenging task. On the one hand we
face rapidly growing resistance towards antibiotics and on the other hand we have a
lack of new antimicrobial substances [1]. Global health care associations consider mul-
tiresistant germs as a global threat, with a special focus on methicillin-resistant
Staphylococcus aureus (MRSA). This germ has a very high prevalence in countries
regardless the medical standard [2]. Infections with MRSA kill nearly 19,000 hospital-
ized patients in the U.S. annually, which is similar to the number of deaths caused by
AIDS, tuberculosis and viral hepatitis combined [3]. Furthermore, infections with
drug-resistant pathogens do increase death, illness, and direct costs by 30100% [4].
Especially, on chronic wounds, pathogenic bacteria can contribute to impaired healing.
Even colonization can lead to non-healing ulcers. Chronic wounds of the lower leg
have prevalence higher than 1% of the population in developed countries. These
wounds do cost billions for Health Care Systems. In European countries they are esti-
mated to account for 12% of the annual health care budget [5]. Venous ulcers are in
general a tedious task to get healed. On average they need 24 weeks to heal, 15% never
heal, and recurrence is found once or in multiple times in 1571% of all cases [6, 7].
In 2003 there was a very interesting finding of plasma physicists who achieved to
generate cold atmospheric plasmas (CAPs) with antibacterial properties. This new
generation of cold atmospheric plasmas shares the same benefits as their hot brothers,
except the enormous heat production. High-temperature plasmas are generally used in
standardized procedures to sterilize equipment or to remove, cauterize and cut tissues
[810]. CAPs have the great advantage of the possibility to provide in-vivo applica-
tions without harming surrounding tissue by heating, since they operate usually below
40C. They have not only bactericidal, but also fungicidal and virucidal properties due
to a highly reactive mix of reactive species, charging reactions, ultraviolet radiation,
and optical and infrared emissions. All the plasma characteristics can be tuned due to
the mechanism plasma is generated (e.g. microwave frequency, radio frequency, high
voltages) and due to the surrounding gas mixtures and concentrations. The great
potential and the broad flexibility to generate CAPs resulted in a rapid, world-wide
ever-growing number of scientists exploring the field of CAPs. Ironically, this trend
shows some similarities to pandemics. The next decades will tell us if CAPs can
answer the high expectations to a broad range of medical indications and others like
in hygiene, bio-decontamination, food security, and other new emerging fields.
In 2005 the Max Planck Institute for Extraterrestrial Physics in Garching (Germany)
developed an indirect argon CAP device, called MicroPlaSter alpha, driven by
microwave technology (2.46 GHz, 86 W, Ar 2.2 slm, distance to the wound 2 cm)
24 Cold Atmospheric Plasma for Clinical Purposes: Promising Results 313
Fig. 24.1 Highly significant higher reduction in bacterial count (~34%, P < 106) in lasma-treated
area (dark grey bar, bottom) compared with standard wound care alone (light grey bar, top)
infrared emissions. The exact bactericidal mechanism is yet unknown and a lot of
effort is done to encode the mode of action. Nevertheless reactive species seem to
play a major role.
In total, we treated over 220 patients in more than 2,000 applications (together
with the department of dermatology, University of Regensburg) using this micro-
wave plasma device and its successor (MicroPlaSter , see Fig. 24.3, which changed
in design and has an additional placebo leg where inert argon gas with the same
temperature as used for plasma treatment can be applied) without having any adverse
effects like pain, yet. Until now, it is unclear whether plasma treatment enhances
wound healing itself. Since wound healing is a long lasting procedure special
designed and controlled clinical trials in patients are needed. Nevertheless there are
some in-vitro or animal studies which support a dosage dependant beneficial effect
of plasma on wound healing and there is data from a NO rich air plasma device from
Russia.
Landsberg and co-workers [13] reported about a positive influence of indirect
DBD on cell regeneration of human keratinocytes (HaCaT). In their study they used
an in vitro wound model (scratch assay). Scratch assay is an established model to
compare closure times. They measured a significantly enhanced wound closure
after plasma treatment compared to untreated control.
Another approach was used of Topala and co-workers [14]. They treated defined
burn wounds on Whistar rats with a daily treatment of a helium plasma jet for 40s.
Even if they detected elevated oxidative stress (markers used: malondialdehyde,
reduced glutathion, glutathione peroxidase, and superoxide dismutase and catalase) a
possible inducer of cell necrosis in plasma treated rats compared to controls.
Interestingly, wound healing in defined plasma dosages was enhanced as proven by
planimetric and histological findings.
24 Cold Atmospheric Plasma for Clinical Purposes: Promising Results 315
Fig. 24.3 Plasma-treatment of chronic ulcer of the left leg using MicroPlaSter -device
But the applied dosage and plasma device play an important role. Keidar and
co-workers [15] reported about the importance of the applied plasma dosage. Mild
helium plasma jet treatments resulted in a reduction of migration rate by factor of 2.
Medium levels lead to cell detachment and intensive treatment to cell desiccation.
The changes in migration rate were explained by plasma-affected integrin expres-
sion. The same group investigated the correlation between tertiary mouse fibroblast
migration rate and the influence of direct plasma jet application [16]. Hundreds of
helium plasma jet application resulted in significant reduced migration rate, whereas
500 s resulted in an even lower migration rate. The authors concluded that as appli-
cation time increases, cells migrate slower. Total reduction of migration rate was
approximately 30%. But it is important that the zone of reduced cell migration cor-
related to the 5 mm direct treatment zone of the jet. This may have a direct influence
on migration rate if directly applied plasma dosage was too high for the cells.
The only data providing evidence of enhanced wound healing due to exogenic NO
rich air plasma comes from a Russian device called Plazon. Many patients were treated
in different clinical trials in Russia. Publications in western journals or online libraries
with this device are not available; same as information about levels of UV (especially
UV-C) or other safety parameter are not published but presented data are indeed
impressive. A clinical trial proved its benefit in 318 patients with venous and arterial
ulcers. Reduction in planimetric determined wound size was significantly decreased
in NO-treated wounds (1.7% per day compared to 0.7% in control group) [17, 18].
316 G. Isbary
Patients suffered from wounds with sizes varying from 6 to 200 cm2. Plasma was
applied from 10 to 30 days (NO concentration ranged from 300 to 500 ppm).
Appearance of granulation and boundary epithelisation, cleansing of ulcers from
exudates and necrosis were much better and faster in NO-treated wounds. Acceleration
factor of 2.5 was stated by authors. Complete healing was achieved 2.54 times
faster than in control leg without plasma. Generally large wounds responded better to
NO therapy. Another trial investigated the effect on diabetic foot ulcers who did not
respond under conventional therapy regimes for 2 months. Those hard-to-heal
wounds responded as well to Plazon therapy [19]. Complete epithelisation was
achieved in small wounds within 68 treatments. Hospitalization time was reduced
by a factor of 2.3 and amputation rate decreased 1.9 times.
Fig. 24.4 Appearance of right groin after 4 additional plasma treatments (left) and after a total of
11 plasma treatments, leaving only residual erythema remaining (right)
24.4 Conclusions
Cold atmospheric plasma technology demonstrated its efficacy not only in in-
vitro but also in the treatment of infected wounds in patients. Our studies prove
the first time, that an additional plasma treatment beside standard wound care can
enhance the reduction of germs on chronic infected wounds significantly. This can
be achieved either by a 5 min plasma treatment, or by a 2 min application.
Furthermore, the study showed in vivo, that plasma reduces different types of
germs, even the multiresistant ones, like MRSA. Therefore, cold atmospheric
plasmas are promising tools for superficial antibacterial applications on infected
wounds in future. There is no evidence that bacteria can become resistant to this
highly reactive mixture of reactive oxygen, hydrogen and nitrogen species, charg-
ing reactions, ultraviolet radiation and optical and infrared emissions. Another
important finding is, that no side effects occurred until now after more than 2,000
plasma applications yet.
The technology is not confined to chronic infected wounds any more. We
demonstrated, that a patient with Hailey-Hailey disease profited by an addi-
tional 5 min plasma treatment, as well. The treated lesions failed to heal with
topical disinfectant and glucocorticoid therapy for many years. Within few
plasma treatments lesions healed, leaving only residual erythema. Burning sen-
sations stopped.
New generations of cold atmospheric plasmas evolve bit by bit. These devices
open the field to completely novel indications, especially for hygiene. One example
is a cold atmospheric plasma which uses a technology called surface micro dis-
charge. It has not only the advantage of a very flexible and freely scalable design
using ambient surrounding air but also the cheap construction costs. First unpub-
lished results are very promising regarding tolerance to the skin besides a highly
antimicrobial potential towards bacteria, spores and even viruses. This technique is
only one example of many others.
However, the exact bactericidal mechanisms of cold atmospheric plasmas are not
understood and a lot of effort has to be done to get this knowledge. This awareness
318 G. Isbary
can help us to enhance future cold atmospheric plasma technologies to have even
more potent devices in the never ending battle against bacterial infections.
Like in other technical developments we are still in the starting blocks of this
innovative scientific leg but the future is in our hands and well see if we can meet
the high expectations.
References
1. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: confronting the
challenges of antibacterial discovery. Nat Rev Drug Discov 6:2940
2. Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E (2006) Emergence and resurgence
of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet 368:874885
3. Klevens RM, Edwards JR, Richards CL, Horan TC, Gaynes RP, Pollock DA, Cardo DM
(2007) Estimating health care-associated infections and deaths in U.S. Hospitals, 2002. CDC
Public Health Report March-April 122:160166
4. Cosgrove SE, Carmeli Y (2003) The impact of antimicrobial resistance on health and economic
outcomes. Clin Infect Dis 36:14331437
5. Etufugh CN, Phillips TJ (2007) Venous ulcers. Clin Dermatol 25:121130
6. Kurz X, Kahn SR, Abenhaim L, Clement D, Norgren L, Baccaglini U, Berard A, Cooke JP,
Cornu-Thenard A, Depairon M, Dormandy JA, Durand-Zaleski I, Fowkes GR, Lamping DL,
Partsch H, Scurr JH, Zuccarelli F (1999) VEINES task force report. Int Angiol 18(2):83102
7. Heit JA (2002) Venous thromboembolism epidemiology. Semin Thromb Hemost 28(2):313
8. Bogle MA, Arndt KA, Dover JS (2007) Evaluation of plasma skin regeneration technology in
low-energy full-facial rejuvenation. Arch Dermatol 143:168174
9. Elsaie ML, Kammer JN (2008) Evaluation of plasma skin regeneration technology for cutane-
ous remodeling. J Cosmet Dermatol 7:309311
10. Kilmer S, Semchyshyn N, Shah G, Fitzpatrick R (2007) A pilot study on the use of a plasma
skin regeneration device (Portrait: PSR3) in full facial rejuvenation procedures. Lasers Med
Sci 22:101109
11. Shimizu T, Steffes B, Pompl R, Jamitzky F, Bunk W, Ramrath K, Georgi M, Stolz W, Schmidt
HU, Urayama T, Fuji S, Morfill G (2008) Characterization of microwave plasma torch for
decontamination. Plasma Process Polym 6:577582
12. Isbary G, Morfill G, Schmidt HU, Georgi M, Ramrath K, Heinlin J, Karrer S, Landthaler M,
Shimizu T, Steffes B, Bunk W, Monetti R, Zimmermann JL, Pompl R, Stolz W (2010) A first
prospective randomized controlled trial to decrease bacterial load using cold atmospheric
argon plasma on chronic wounds in patients. Br J Dermatol 163:7882
13. Landsberg K, Neumann M, Haehnel M, Scharf C, Weltmann KD, von Woedtke T (2010)
Atmospheric pressure plasma shows rapid regeneration of human cells. In: Proceedings of 3rd
international conference on plasma medicine, Greifswald, Germany, 1924 Sept
14. Topala I, Nastuta AV, Grigoras C, Dumitrascu N (2010) Study of oxidative stress markers dur-
ing epithelial regeneration induced by atmospheric pressure plasma treatment. In: Proceedings
of 3rd international conference on plasma medicine, Greifswald, Germany, 1924 Sept
15. Keidar M, Volotskova O, Shashurin A, Stepp MA (2010) Plasma control of cell migration.
In: Proceedings of 3rd international conference on plasma medicine, Greifswald, Germany,
1924 Sept
16. Volotskova O, Shashurin A, Stepp MA, Pal-Ghosh S, Keidar M (2010) Plasma-controlled cell
migration: localization of cold plasma-cell interaction region. Plasma Med 1:8592
17. Shekhter AB, Kabisov RK, Pekshee AV (1998) Byull Eksp Biol Med 8:210
18. Shekhter AB, Serezhenkov VA, Rudenko TG, Pekshev AV, Vanin AF (2005) Nitric Oxide Biol
Chem 12:210
24 Cold Atmospheric Plasma for Clinical Purposes: Promising Results 319
Abstract The assumption is that tissue tolerable plasma works as promoter for
wound healing and can be beneficially combined with the antiseptic polihexanide to
avoid bacterial recolonization. The effects of a combined plasma polihexanide
(PHMB) application on cell integrity, cytotoxicity and its irritative and inflamma-
tive potential were tested in vitro and in two dogs in vivo.
25.1 Introduction
Two important characteristics of a chronic wound beside other reasons are chronic
inflammation and critical bacterial colonization. The latter is one of the main
reasons to impede the healing process. Only a few of planctonic bacteria are
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 321
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_25, Springer Science+Business Media B.V. 2012
322 C.P. Bender et al.
25.2 Plasma
The plasma was generated with the atmospheric pressure plasma jet (Neoplas
GmbH, Greifswald, Germany, CE certification No. 609.003.1) (Fig. 25.2) as
described in Weltmann et al. 2009 [13], with argon being used as a carrier gas.
The gas flow was 5 slm per min. The plasma was used in continuous mode.
25 Tissue Tolerable Plasma and Polihexanide 323
The visible plasma tip was 11 mm from the nozzle; the thermal output was 160 mV,
the input power 3.9 W.
Fig. 25.2 Left: Schematic of the atmospheric pressure plasma jet. Right: Treatment of reconstructed
human epidermis with the atmospheric pressure plasma jet
To monitor the integrity and barrier function of the reconstructed human epidermis
the transepithelial electrical resistance was measured before and after treatment
with the EVOM/STX2 (WPI Germany, Berlin, Germany).
The 10s plasma treatment led to a very little decrease of the TEER, whereas the
20s plasma treatment led to approximately 50% decrease of the TEER. This gives
an advice of the affected integrity of the keratinocytes and that the barrier function
of the epidermis is reduced. But compared to the chemical corrosive standard Triton
X 1%, the reduction of the barrier function is moderate. A possible dose effect
relation of plasma impact is visible (Fig. 25.3).
25.3.2 Viability-MTT-Assay
4.5
TEER before treatment
TEER 4
[kOhm] TEER after treatment
3.5
2.5
1.5
0.5
0
50l TXT1%, 2h 50l BSS, 2h Gas-control 20s Plasma 10s Plasma 20s
Fig. 25.3 Transepithelial Electrical Resistance (TEER) before and after (i) 2 h contact time of
50 ml Triton X 100, 1% (50 ml TTX 1%, 2 h) as positive corrosive control, (ii) 2 h contact time of
50 ml Balanced Salt Solution (50 ml BSS, 2 h) as negative control, (iii) 20s argon gas treatment
(Gas-control 20s), (iv) 10s and 20s plasma treatment; Error bars: confidence intervals, n = 6,
alpha = 0.05
120,00
100,00
80,00
viability [%]
60,00
40,00
20,00
0,00
50l BSS, 2h
300l, 30min
300l, 30min
TTX,1%,2h
Gas, 20s
Gas, 20s +
Gas, 20s +
Plasma, 10s
Plasma,10s +
Plasma, 10s +
Plasma, 20s
Plasma, 20s +
Plasma, 20s +
Lavanid 2
Lavanid 1
Lavanid 1,
Lavanid 2,
Lavanid 2
Lavanid 1
Lavanid 2
Lavanid 1
50l
Fig. 25.4 Viability (%) of the reconstructed human epidermis after treatment with (i) 50 ml Triton
x-100, 1%, (ii) 50 ml BSS, (iii) Lavanid (1 and 2), (iv) Argon gas 20s, (v) Argon gas+Lavanid
(1 and 2), (vi) plasma (10s and 20s) and (vii) combined treatment plasma/polihexanide
(10s+Lavanid1, 10s+Lavanid2, 20s+Lavanid1, 20s+Lavanid2). Error bars: confidence intervals;
n = 6, alpha = 0.05
After the combined treatment, any liquid remaining on the reconstructed human
epidermis was removed. The epidermis comprising cell-culture-inserts were then
placed into 24-well plates containing 300 ml MTT solutions per well and incubated for
3 h in a 5% CO2 incubator at 37C. Then they were immersed in 2 ml of acidified
Ethanol as extractant solution over night at 8C. The absorbances of 200 ml extractant
solutions were measured in 96-well microtiter plates at 560 nm. The test was evaluated
as% viability compared to the negative control (2 h contact time BSS) (Fig. 25.4).
326 C.P. Bender et al.
10
6
directly
SSM 5 after 5min
after 24h
4
0
300l Lavanid1 plasma10s+300l Lavanid1 plasma 10s gas10s+300l Lavanid1
Fig. 25.5 Score sum mean (SSM) of the chorioallantoic membranes (each group n = 6),
immediately after treatment, after 5 min and after 24 h
towards sensitive tissues like wounds is described in Table 25.1. The allocation is
based on the reaction of the CAMs in line with Kramer and Behrens-Baumann
(1997) [17]. Five different and clearly visible tolerance categories can be distin-
guished. Allocating the SSM, we tried to take this classification into account.
Single treatment with polihexanide 0.02% (Lavanid 1) led to a SSM of 8.7 within
24 h, what is a well compatibility (Table 25.1). Also single plasma treatment had a
similar compatibility and led to a SSM of 8.8. The combined treatment led to a
slight worsening of the compatibility, but the SSM of 9.2 still implies a moderate
compatibility. There was no evident difference to the combined treatment of gas/
polihexanide (SSM 8.2). In comparison to the previous study [5] the plasma effects
in this study are weaker because of the increased distance from the nozzle to the
CAM, (10s plasma after 24 h: 8.8 [here] versus 12.89 [5]).
Following these results and the results of earlier studies [5, 6] it was shown that
the plasma compatibility is dependent on several different parameters as flow rate,
distance, power, treatment time and treatment mode. The combined treatment of
TTP and polihexanide in this study led to a very good compatibility in the HET-
CAM within 5 min. Because of the good predictivity of the HET-CAM for non-
irritants in vivo [18] it is probable that a combined treatment is tolerable for wounds
328 C.P. Bender et al.
using the tested settings. The HET-CAM provides a good model to determine
compatible parameters for plasma treatments of eyes and wounds.
The positive results from the in vitro assays encouraged us to test the combined
treatment in two dogs. Both dogs suffered of chronic wounds for several years,
conventional therapies (surgical debridement and wound closure in case of the
chronic rhagade, self adhesive dressing and different ointments several times a
week, prescribed by a veterinarian and applied by the owner in both cases, protec-
tive bandages in the second case) failed. The treatment was well tolerated by both
animals. In one case, the treatment led to a complete healing after 11 weeks,
which was particularly remarkable because the treatment with TPP or polihexanide
alone did not led to healing. This supports the presumption, that TPP and poli-
hexanide have synergistic effects in promoting healing of chronic wounds. In the
second case, the treatment was complicated by constant licking of the wound by
the dog, but the healing progressed after applying a ruff and the wound has
improved since then.
The first patient was a 9-year-old German shepherd dog with a chronic rhagade of
the nasal planum. It can be assumed that the nose was regularly exposed to bacteria
because the dog licked his nose several times a day and rummaged in the soil some-
times. Swabs were not taken. The wound had persisted for 4 years and was pre-
treated with 0.02% polihexanide for a period of 17 months leading to a wound
surface reduction by approximately 30% of the original wound surface. Subsequently,
the wound healing process stagnated.
The wound was experimentally treated by applying TTP combined with 0.02%
polihexanide solution for two weeks. A slight amelioration was visible. After an
interruption of 3 weeks (break through holidays), the therapy was continued apply-
ing exclusively TTP for a period of 11 weeks to find out, if a sole plasma treatment
would lead to healing. The dog was subjected to the plasma treatment in two ses-
sions of 15s, each, two or three times a week. During the treatment, the tip of the
visible plasma contacted the vital tissue (distance to the nozzle about 11 mm, 5 slm
argon gas, derived from [4]) (Fig. 25.6). The plasma jet was moved quickly over the
wound surface on a meandering course. Consequently, the complete wound surface
was in contact with the plasma several times. The sole plasma therapy was finished
because there was no visible progress in the healing process. After the plasma treat-
ment was finished, the wound was treated with polihexanide daily until the wound
finally closed after a further 11 weeks (Fig. 25.6).
25 Tissue Tolerable Plasma and Polihexanide 329
Fig. 25.6 Plasma treatment (top left), state at the treatment start (top right) and healing progress
after 6.5 weeks (down left) and 11 weeks (down right)
The second patient was a 12-year-old male German shepherd dog with a chronic
wound on the left dorsal carpus (foreleg), occurred after a trauma, caused by a
barbed wire fence 3 years ago. The wound was treated several times by a veterinary
with different ointments and protective bandages without any success.
Before starting the treatment, a swab was taken from the wound and mass of
haemolytic staphylococci, greening Streptococci and sporformers were found. The
wound was experimentally treated by applying TTP exclusively for two weeks
(Fig. 25.7). Due to the bigger size of the wound the treatment time was prolonged
to 90s on a meandering course, two times a week. Because the dog did not stop lick-
ing, the plasma treatment was continued in combination with daily treatment of
0.02% polihexanide for another 2 weeks. After an interruption of two weeks to
observe if the wound healing occurs, the therapy was continued because the con-
tinuative licking impeded the healing progress and a beginning of exsudation
330 C.P. Bender et al.
Fig. 25.7 Plasma treatment (top left), state at the treatment start (top right) and healing progress
after 3 weeks (down left) and 8 weeks (down right)
became visibly. The combined therapy was continued for another nine sessions, two
plasma applications per week. The healing progressed after applying a ruff and the
wound has improved since then (Fig. 25.7). After the plasma treatment had finished,
the wound was treated with polihexanide daily until the wound finally closed after
a further 8 weeks.
25 Tissue Tolerable Plasma and Polihexanide 331
In the first case, when the plasma treatment was stopped and polihexanide was
applied again, the wound, which had persisted for 4 years, continuously healed and
closed completely within 11 weeks. Accordingly, the wound healing supposedly
progressed as follows: The initially sole polihexanide treatment stimulated the
wound healing simultaneously repelling the critical colonization so that the wound
grew smaller by about one-third. Thereafter, healing of the wound stagnated. The
combined application of polihexanide and TTP followed by sole treatment with
TTP revived the wound healing process. During this time, however, the process had
obviously changed significantly so that the wound after the discontinuance of the
TTP application clinically changed to become a wound progressively healing until
complete closure. This effect is probably attributable to the TTP application. When
plasma was applied in the modified HET-CAM point by point in the range of sec-
onds, the following responses could be observed: hyperemia, hemorrhage and, after
24 h, contraction, granuloma formation with spoke wheel-like angiogenesis and
extravasale coagulation. It can be assumed that similar processes occur in vital tis-
sue of mammals. Accordingly, the plasma treatment should induce a variety of
effects causing the wound to transform from the chronic into the acute phase, for
instance by possible stimulation of the fibroblasts and keratinocytes, angiogenesis,
contraction and coagulation. The TTPs capability of tissue alteration induced
inflammation and improved blood circulation produced further stimuli for wound
healing processes [19] most likely as a summation of the physico-chemical plasma
components [20]. The healing effect of TTP could be explained by generation of
reactive oxygen species (ROS) during TTP treatment, which have a significant
molecular effect on the stimulation of wound healing. ROS can directly stimulate
MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF
[heparin-binding EGF (epidermal growth factor)]. Additionally, ROS can induce
tyrosine phosphorylation of PDGF (Platelet Derived Growth Factor) alpha- and
beta-receptors. Binding of both growth factors to its affinity receptor elicits a variety
of cellular responses, e.g. MAPK (mitogen-activated protein kinase) activation fol-
lowed by an induction of transcription. It is released by platelets upon wounding
and plays an important role in stimulating the growth of adjacent cells and thereby
healing the wound [2124]. Nevertheless, all affecting impact factors are dose
dependent. Further investigations should, therefore, be aimed at elucidating the cel-
lular mechanisms of the plasma effects on wound healing.
Positive possible side effects are the antiseptic action of TTP [6, 7] and its capa-
bility of inactivating biofilms [8, 9]. This is particularly important for the nasal
wound to be treated as the regular licking in the area of the injured nasal planum
involves an increased entry of bacteria into the wound [2527]. The antiseptic treat-
ment with polihexanide, which had been continued after the plasma treatment, had
the effect that a new critical colonization and biofilm formation could be avoided.
Contrary to an experts recommendation [28] we applied polihexanide for an
extended period of time, so as to avoid the risk of biofilm formation in the case of
the dog licking its nose.
332 C.P. Bender et al.
It can be derived from the wound healing process that the TTP induces an
inflammation, which is possibly associated with a cellular stimulation as probably
visible in the MTT-assay. The inflammatory response transforms the chronic inflam-
mation into an acute one, the further progress of which is stimulated by healing
effects including that of polihexanide. To date, the time sequences in which these
effects occur are still unknown. As a result, the secondary wound healing did not
progress any further when the wound was treated with TTP two or three times a
week, implying that inflammatory effects are probably predominating. After the
TTP treatment was terminated the second stage of clinical visible healing began and
the wound finally closed completely. The application of polihexanide at this stage
prevented the formation of new biofilms and a disturbance of the wound healing.
In the second case, the licking surely will be a major reason to retard the wound
healing. The dog licked his wound even before the plasma treatment. The reason
for the licking seems unclear - it could be itching and/or pain and/or a bad habit.
Nevertheless, the wound closure failured when protective bandages and ointments
were applied in previous therapies. Therefore, it can be assumed that other reasons
among the licking as critical bacterial colonization and chronic inflammation led to
a disturbance in the wound healing process. With the exclusion of licking, the
combined plasma therapie led to healing, what was not achieved in earlier
treatments.
It is notable that the TTP was well tolerated by the animals. Thus, painful surgery
or anesthesia for such intervention could be avoided, what is a huge advantage in
anaesthesia risk patients.
Chronic wounds in animal patients are not standardized, what makes comparing
difficult. In these two cases, it lacks of knowledge about the optimum treatment
times and frequencies. The in vitro tests give advices for the maximum single
treatment, but it is still unknown what are the optimal treatment intervals and
frequencies. Further investigations are still necessary.
25.6 Conclusion
To conclude, the applied in vitro methods (modified HET-CAM for irritation - and
inflammation potential; reconstructed human epidermis for the assessment of the
cytotoxicity and integrity) are useful for screening plasma sources for their suitabil-
ity for chronic wound treatment and to determine the parameters for medical appli-
cations alone and in combination with polihexanide.
The in vivo TPP applications have been tolerated by the dogs without pain.
On one hand, the different treatment regimes and the small number of cases
allow no strong conclusion with regard to synergistic TPP-polihexanide effects. On
the other hand, the final success of the combined treatment supports the hypothesis
that the combined TPP-polihexanide treatment has possible synergistic effects,
particular in regard to the fact that the initially sole polihexanid treatment in the first
case led not to healing. The combined TPP-polihexanide treatment could be a
25 Tissue Tolerable Plasma and Polihexanide 333
promising option for the treatment of chronic wounds. But as only a very little
amount of experience in the plasma application on chronic wounds exists, further
investigations should, therefore, be aimed at elucidating the cellular mechanism of
plasma promoted wound healing, combined TPP-polihexanide application and
optimizing the course of treatment.
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334 C.P. Bender et al.
Abstract A new field of plasma applications developed in the last years, entitled
plasma medicine, has focused the attention of many peoples from plasma community
on biology and medicine. Subjects that involve plasma physics and technology
(e.g. living tissue treatment or wound healing, cancer cell apoptosis, blood coagula-
tion, sterilization and decontamination) are nowadays in study in many laboratories.
In this paper we present results on optical and electrical diagnosis of a helium
atmospheric pressure plasma jet designed for medical use. This type of plasma jet was
used for improvement of the wound healing process. We observed a more rapid mac-
roscopic healing of the plasma treated wounds in comparison with the control group.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 335
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_26, Springer Science+Business Media B.V. 2012
336 I. Topala and A. Nastuta
The atmospheric pressure plasma jet (APPJ) designed in our laboratory is based on
a cylindrical barrier discharge with external electrodes. Various kinds of geometries
are reported in the literature to generate dielectric barrier discharge (DBD) plasma.
Arc regime transition is avoided due to the presence in the electrode gap of dielec-
tric layer. This also assures the current limitation and constraints the system to work
in pulsed mode. Two working regimes are known: filamentary discharge (random
streamers) and homogenous discharge (diffuse or glow) [19, 20].
We have used a quartz tube, 4 mm inner diameter and 6 mm outer diameter, to
generate an atmospheric pressure plasma jet. Aluminum tape electrodes (10 mm
width for the power electrode and 4 mm width for the ground electrode) are wrapped
on the external surface of the tube, being separated by a 10 mm gap. This solution
offers a high experimental flexibility. The set-up can be easily moved and adapted
to work in biological environment, or can be integrated in other experimental
arrangements to perform in-situ analyses of plasma or its effects on biological mate-
rial. Helium (spectral purity, 99.99999%) is flowing inside the quartz tube with
electronically controlled flow rate. For flow rate values smaller than 7 L/min a lami-
nar flow regime is assured. The residence time of helium atoms in the electrode gap
has values up to 15 ms. The speed of helium atoms increases linearly from 1.4 m/s
at 1 L/min to 14 m/s at 10 L/min. The use of helium as working gas can be consid-
ered expensive for many applications. However for medical applications this is not
necessarily true, since the typical treatment durations are explored in the seconds to
only few minutes range.
The analysis of the discharge current shape proves that our discharge works in a
homogenous regime. The current is characterized by a sharp peak during the increase
of the voltage pulse, when the ground electrode is acting as cathode and the powered
electrode as anode. This is called usually the primary discharge. Unique for the
DBD set-up is the appearance of a secondary discharge during the falling part of the
applied voltage pulse, the above mentioned roles of the electrodes being now
26 Helium Atmospheric Pressure Plasma Jet: Diagnostics and Application 337
Fig. 26.1 Influence of voltage pulse (a) amplitude on main parameters of the discharge: current
(b), power (c), charge and current pulse duration (d), measured as full width at the half maximum
(frequency 2 kHz, helium flow rate 3 L/min)
inverted (Fig. 26.1a). The necessary energy to develop this second discharge is
assured by the charges deposited onto the dielectrics surface during the primary
discharge [21].
The value of the discharge current peak for these two discharges separated in
time is controlled mainly by the applied voltage amplitude (Fig. 26.1b). These low
values, in the mA range, assure the safety use of this plasma jet in direct contact
with living animals without any perceptible sensory or motor response. This was
tested in our laboratory on Wistar rats back skin and on human fingers.
The power peak values, obtained as the product of the primary current peak and
the corresponding value of the instantaneous voltage, is increasing together with the
increasing of the voltage (Fig. 26.1c). The same remark is valid for the transported
charge between the electrodes. Due to the sharpening of the primary current peak
(Fig. 26.1d), the average value of the pulse power slowly increases in the range
12.5 W (Fig. 26.1c).
The values of the electron density, obtained from both experiments and simula-
tions, in helium atmospheric pressure glow discharge are situated in the range 1010
1012 cm3 [19, 22, 23].
Utilization of plasma in direct contact with living animals must take into
account not only the charge effects, but also the presence UV radiations and
338 I. Topala and A. Nastuta
chemical active species, that can be related to plasma toxicity. Last but not the
least, local temperature or pH modifications in biological supramolecular sys-
tems can induce denaturation or destruction of molecules or supramolecular
assemblies.
The emission spectrum of the discharge contains helium lines, centered at 388.1,
501.5, 587.5, 667.8, 706.5, 728.1 nm. Lines or bands of impurities from the air were
identified in the APPJ emission spectrum at the following wavelengths: O (777.4 nm),
N2+ (391.4, 427.8, 470 nm), N2 (315.9, 337.1, 357.6, 380.4 nm), OH (308.9 nm), H
(486.1, 656.3 nm). No emission was found in the 200300 nm range. The time aver-
aged emission intensity increases with the voltage and for a fixed value of the volt-
age the maximum value of the emission intensity was found between the electrodes.
For all mentioned excited species, excepting the molecular nitrogen, lower emission
intensity was found outside the quartz tube, i.e. in the jet region.
The concentration values of the oxygen and nitrogen reactive species generated
in the APPJ are presented in the Table 26.1.
Plasma generation of UV radiations is a major concern regarding the medical use
of plasma based devices. An exception is obviously the use of plasma for steriliza-
tion and decontamination, where UV radiations were found to be a key factor for an
efficient process [1, 2729]. Nevertheless, depending on the operating conditions,
the effect of UV photons in the sterilization process can be neglected [30]. Typical
dose values for the plasma jets or other plasma sources designed for living tissue
treatment were found to be less than the necessary dose to induce skin or cell dam-
age [26, 31, 32].
The temperature of our helium APPJ was estimated by various methods. First of
all the rotational temperature of nitrogen molecular ion was estimated using the
Boltzmann plot method [33]. Between the electrodes the temperature values are
around 300 K, while in the jets tip region these values reach at 700 K. This is
clearly an overestimation of the gas temperature value. If we focus the plasma jet on
a laboratory thermocouple (situated at 15 mm from the edge of the quartz tube) we
obtain values of 296.5 K at 4 kV, 299.5 K at 6 kV and 304.5 K at 8 kV. Moreover,
pictures obtained with laboratory infrared camera of the plasma jet prove that the
plasma jet has an overall temperature equal to the background gas in the room
(Fig. 26.2).
Fig. 26.2 Visible and infrared picture of the helium APPJ in contact with a human finger (voltage
pulse amplitude 6 kV)
Time and space propagation of the plasma jet from the production region to the liv-
ing tissues is of a special interest. Most of the plasma sources designed for medical
use are pulsed discharges, the frequency has values in the kHz range up to micro-
wave range. This leads to an exposure of living tissues to UV-visible photons pulses
and to pulsed free radical chemistry.
In order to study the appearance and propagation of the helium APPJ (voltage
pulse amplitude 6 kV), we have used the high speed photography technique.
Pictures of the APPJ were taken for the plasma jet focused on a quartz window,
placed at 15 mm from the tubes edge and for the plasma jet facing a human finger,
placed at the same 15 mm distance from the tubes edge (Fig. 26.2). The time t = 0
was considered the moment when the discharge current corresponding to the pri-
mary discharge starts to increase.
As already reported in the literature, the jet is not spatially homogenous during the
current peak, during both primary and secondary discharges. A closer look in the
nanoseconds range as exposure time reveals the existence of plasma structures, so-
called plasma bullets, which can be found in distinct regions as the time increases. The
plasma propagation mechanism in air can be explained using a streamer model based
on photo ionization [35, 36] or using an ionization wave model [37]; new hypotheses
are under investigation and even glow discharge like behavior can be obtained [38].
During the primary discharge, inside the quartz tube the discharge appears as
homogenous luminous structure, in the anodes region and heading towards the cath-
ode. A discharge channel is then formed in the center of the quartz tube. After the
expansion in the air, the plasma bullets are no longer homogenous, a ring-shape
340 I. Topala and A. Nastuta
Fig. 26.3 Typical pictures of the APPJ behavior in on the surface of a quartz substrate (side view
and on-axis view) and a human finger (side view); temporal behavior of the plasma light intensity
at the human finger surface, as obtained from the analysis of ICCD images
profile of the emission intensity being recorded due to the higher density of molec-
ular nitrogen in the plasma volume [3941]. If the helium APPJ is focused on a
substrate the plasma bullet spreads on the surface and then is extinguish. We found
a different behavior of plasma structures dynamics onto dielectric substrates (i.e.
the quartz window) and onto human tissues (Fig. 26.3). In the space between the
tubes edge and the substrate the plasma jet has a bullet like behavior. Then the
plasma is reaching the substrate and it starts to spread. For a specific duration a
plasma structure exists in the front of the substrate. This duration is very short for
the quartz substrate, of around 1 ms and is very long for the human finger, of about
20 ms (Fig. 26.3).
Upon our knowledge, these are the first ICCD investigations of the plasma jet
action on a living human tissue. It should be emphasized that a long living
plasma structure exists at the human finger surface, in comparison with plasma
26 Helium Atmospheric Pressure Plasma Jet: Diagnostics and Application 341
Many studies revealed the great potential of plasma in the treatment of skin wounds,
with an especial attention devoted to chronic infected wounds. Indirect or direct
action of plasma on wounds was found to induce the healing of the injured tissues
[4245].
Our study was focused on plasma treatment of fresh model wounds obtained on
the Wistar rats back skin by chemical burns. The exposure of the rats skin to a
sulphuric acid solution caused a second degree burn, both the superficial dermis
and the epidermis being damaged. Control wounds were selected to study the natu-
ral wound regeneration, while other wounds were used to study the influence of the
plasma treatment on the regeneration process. These wounds were exposed 40 s
daily to the helium plasma jet, without any preliminary local cleaning or wound
conditioning. Hematological, biochemical and histological data were monitored
over the observation period (21 days) in order to follow the evolution of systemic
and local effects. Experimental procedures are described in detail in our previous
publications [33, 46, 47]. The experiments were carried out together with our col-
leagues from the Gr.T. Popa University of Medicine and Pharmacy, Physiopathology
Department, Iasi, Romania, under the supervision of Prof. Dr. Magda Badescu.
The values of selected hematological and biochemical parameters at day 21 are
as follows: white blood cell, 5.8 109 cells/L for control group, 9 109 cells/L for
natural wounds recovery group and 5.6 109 cells/L for plasma treated wounds
group; fibrinogen, 175 mg/dL for control group, 260 mg/dL for natural wounds
recovery group and 185 mg/dL for plasma treated wounds group; C3 component,
100 mg/dL for control group, 210 mg/dL for natural wounds recovery group and
100 mg/dL for plasma treated wounds group.
After 21 days the natural wounds recovery process is not finished. The center of
the initial wounds is still visible. As against this behavior, the recovery process for
the plasma treated wounds appears to be accelerated. The wounds disappeared
almost completely (Fig. 26.4).
Further biochemical analyses of skin homogenates from the wound region,
revealed differences on oxidative stress markers as against control group [33, 46,
47]. Values of measured markers are as follows: malondialdehyde, 0.22 mmol/mL
homogenized skin for control group, 0.48 mmol/mL homogenized skin for natural
wounds recovery group and 0.82 mmol/mL homogenized skin for plasma treated
wounds group; reduced glutathione, 0.82 nmol/mg protein for control group,
0.48 nmol/mg protein for natural wounds recovery group and 0.38 nmol/mg pro-
tein for plasma treated wounds group; glutathione peroxidase, 79 mmol GSSG/
min/mg protein for control group, 64 mmol GSSG/min/mg protein for natural
342 I. Topala and A. Nastuta
wounds recovery group and 31 mmol GSSG/min/mg protein for plasma treated
wounds group; catalase, 16,000 U/mg protein for control group, 10,000 U/mg
protein for natural wounds recovery group and 5,000 U/mg protein for plasma
treated wounds group.
Plasma action was found to affect biological function in cells and tissues, trough
peroxidation of lipids from cell membranes, oxidative modification of proteins
involved in signalling pathways, oxidative DNA damage [4, 48].
To conclude, helium atmospheric pressure plasma jet represents a good option
as a plasma source for medical applications. Its operation is reproducible and can
be easily standardized, while the economic aspects can be adjusted to obtain a
profitable system. The mechanism of plasma action and its effects in biology and
medicine are not yet fully understood [49]. For a better understanding of mecha-
nism and the reaction pathways that are responsible for the benefic effects of
plasma use in medical applications, experiments regarding plasma effects on
supramolecular biological systems like proteins are carried now our group. Plasma
effects on protein structure and the relations between structure and function are
investigated. The results will offer a much clear image of the plasma effects on
biological systems.
Acknowledgements The authors thank to Dr. Constantin Grigoras (Gr.T. Popa University of
Medicine and Pharmacy, Iasi, Romania) for its valuable help in wound healing experiments and for
the fruitfull discussions.
This work was supported by CNCSIS-UEFISCSU, grant PN II-RU 297/2010-2011 and ESF in
Romania, grant POSDRU/89/1.5/S/63663.
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Chapter 27
Non-equilibrium Air Plasma for Wound
Bleeding Control
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 347
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_27, Springer Science+Business Media B.V. 2012
348 S.P. Kuo et al.
surgery for bleeding control [35]. The heat carried by argon plasma, produced by
a high-frequency discharge between the tip of a probe and the target tissue, cauter-
izes and desiccates blood. However, it is difficult to treat larger external wounds. On
the other hand, reactive oxygen metabolites produced in non-equilibrium air plasma
could also induce blood clotting effect. Oxidants can affect several key steps of
platelet function to indirectly enhance platelet agglomeration through local increases
in platelet-activating factor (PAF) [6]. Furthermore, oxidants promote de novo syn-
thesis of tissue factor pro-coagulant activity [7]. Indeed, Kalghatgi et al. [8] showed
that a blood sample could be clotted by direct contact of the sample to non-thermal
atmospheric pressure plasma [9], produced by a dielectric barrier discharge (DBD).
Therefore, new devices that can deliver copious low temperature plasma, which will
not cause thermal damage on the tissue surrounding the wound, would be
desirable.
Chen et al. [10] and Kuo et al. [11] showed that a low temperature non-equilibrium
air plasma [12] could clot anti-coagulated whole blood samples in less than 20 s,
which is much less than 30 min for an untreated sample to reach complete coagula-
tion. It was also found via emission spectroscopy that this plasma spray produces
abundant reactive atomic oxygen (RAO) in its plasma effluent [13]. Activation of
erythrocyte platelet interactions by RAO to trigger blood coagulation was sug-
gested as a plausible coagulation mechanism.
However, those experimental results performed under well-controlled in-vitro
conditions may not describe what occurs in the much more complicated in-vivo
environment. In the present work, we use pigs as the animal model [14] to perform
in-vivo tests of blood coagulation by non-equilibrium air plasma [12]. The effective-
ness of this plasma spray to stop bleeding is studied. The results of these tests are
presented and discussed. Post-operative observation of straight cut and cross
cut wound healing has been conducted. The impact of plasma treatment on wound
healing is reported.
Fig. 27.1 (a) A photo and (b) a schematic of the plasma spray; (c) voltage V, current I, and power
P of the discharge in one cycle; (d) the spatial distribution of the plasma emission intensity at
777.4 nm; the numerical value at each contour is the log10 of the intensity in Rayleigh; (e) a video
graph of the plasma plume
about 12.5 mm above the ring-shaped outer electrode. A photo of the device
with the generated plasma plume and its schematic are shown in Figs. 27.1a, and b,
respectively.
The discharge is periodic at 60 Hz and in each AC cycle there are two discharges.
The ionization percentage of the plasma produced by the discharges is less than
one thousandth of one percentage of the airflow (i.e., <105); the plasma spray gas
is dominated by nitrogen and oxygen molecules. The time varying voltage V(t)
and current I(t) of the discharge were measured using a digital oscilloscope
(Tektronix TDS3012 DPO 100 MHz and 1.25 GS/s), where V is the voltage of the
central electrode of the device (outer electrode is grounded). Current I was
350 S.P. Kuo et al.
measured by a current loop (0.1 V/1A) around the electric wire connected to the
outer electrode. All of the discharge current passes through that wire. The product
of the V and I functions gives the instantaneous power function P(t). The V, I and
P in one cycle are presented in Fig. 27.1c. As shown in Fig. 27.1c, the positive
(PD) and negative (ND) discharges are quite different in their characteristics. ND
is a standard arc discharge with a rapid voltage drop at the start of the discharge;
on the other hand, PD is probably in the arc-glow transition, which has a higher
maintaining voltage to drive larger current and consumes more power. The peak
power in the positive discharge reaches 2 kW; the overall average power is about
350 W.
The RAO flux of the plasma spray was determined via the emission spectroscopy of
the plasma plume [13]. We measured the spatial distribution of the 777.4 nm emis-
sions from the 5P state of atomic oxygen (OI). The image data were taken with an
Apogee Alta E47 thermoelectrically cooled CCD camera with a Nikon 50 mm f/1.2
lens and a 50 mm diameter 2 nm pass band interference filter with a center wave-
length of 777.4 nm. The choice of lens relative to detector size ensures that the tar-
get emission line passes through the filter over the entire field of view, allowing the
camera to measure the entire plasma plume simultaneously. Another advantageous
feature of the camera measurement is that, for a diffuse pixel-filling source such as
the plasma plume, the distance between the camera and the source does not change
the measured intensity per pixel.
The camera was calibrated to determine the bias counts, thermal noise, and sen-
sitivity by acquiring images of an integrating sphere calibration source traceable to
the National Institutes of Standards and Technology (NIST) at the exposure times
and aperture settings used in the experiment. A calibration table supplied with the
source and the filter transmission documented by the manufacturer provides a linear
relationship between pixel counts and the intensity in Rayleighs of 777.4 nm emis-
sions in the plasma plume for each aperture setting and exposure time, where 1
Rayleigh = 1010 photons/m2 s, weighing the apparent emission rate integrated along
a line of sight. The volume emission rate in photons m3 s1 can be determined by
dividing the apparent intensity in Rayleighs by the path length of the emitting
region.
Due to the short lifetime (~0.20.3 ms) of atomic oxygen [15], the axial distri-
bution of the emission intensity varied strongly and to have a sharp upward extent.
To capture the full axial extent of this distribution beyond the dynamic range of a
single 16-bit digital image (which is limited to four order of magnitude), multiple
images of the plasma were recorded separately with a moveable stage approxi-
mately 0.15 m square draped in black velvet cloth blocking the brighter portions of
the plasma effluent to allow operation of the camera at higher sensitivity and lon-
ger exposure times while avoiding saturation in the brighter parts of the plasma
27 Non-equilibrium Air Plasma for Wound Bleeding Control 351
plume. Due to the size of the barrier and the diffuse covering, the measurements
were made in the geometrical optics regime with diffraction negligible relative to
the resolution of the camera.
The imaging process started with the barrier completely lowered, the bright
emissions from the direct flames required the camera to be operated at its lowest
sensitivity, with the aperture stopped to f/16 and exposure time set to 0.1 s. Even at
this shortest exposure time of 0.1 s, the image was an average over 6 discharges. In
other words, the instantaneous fluctuation of the emission in the image due to the
60 Hz alternating current driving the plasma spray has been evened out. The cover
was then moved up by 3 mm sequentially in each image, with the aperture and
exposure time adjusted accordingly at each stage. It proceeded until the cover
blocked most of the flame region to detect the upward boundary of the emission
using the camera at high sensitivity with the aperture opened to f/1.2 and the expo-
sure time set at 1 s. The resulting images were then calibrated and combined into a
composite image giving contours of the intensity distribution, ranging from 105 to
109 Rayleighs, as shown in Fig. 27.1d. An image of the plasma plume of the spray
recorded by a video camera is included in Fig. 27.1e for reference. As shown,
the visible plasma plume extends out axially to about 40 mm from the nozzle of the
device, where the intensity of 777.4 nm emissions is about 105 Rayleigh; i.e., the
total photon emission from a slice of the plasma effluent at 40 mm away from
the nozzle is about 10 15 m 2 s1. Assuming no other emissions are present
within the filter passband, and incorporating the air flow speed of ~20 m/s (on
average at 40 mm away from the nozzle), the corresponding flux and density of 5P
state OI are estimated to be about 6 1013 m2 s1 (i.e., ~1015/[(4p) (4/p)]) and
3 1012 m3, respectively. A spectrometer was also used to scan the emission spec-
trum of the plasma from 300 to 900 nm, in which intensive lines contributed by
oxygen radicals appear also only around 777.4 nm. The UV radiation from 300 nm
to 400 nm was not detected.
There are three likely processes to produce atomic oxygen; one is to dissociate
an oxygen molecule into two oxygen atoms via the reaction e + O2 2O + e, which
has a reaction rate coefficient k1 = 4.2E9exp(5.6/Te) [16], where Te is in eV. This
reaction rate decreases rapidly with Te < 5.6 eV; thus it needs about 5 eV electrons to
effectively dissociate O2 into atomic oxygen. The second one is through recombina-
tion of charged particles e + O+ O, which may not need the presence of energetic
electrons. The third one is the dissociative attachment of electrons to molecular
oxygen e + O2 O + O; conservation of energy requires that the electron energy
exceeds a threshold level, which normally is 3.6 eV. Though this threshold level
decreases as the internal energy of molecular oxygen increases, laboratory experi-
mental results [17] show that this level can go down to 1 eV and less. Therefore, the
third process of dissociative attachment is likely the dominant process of OI genera-
tion by this low temperature plasma spray. The 5P state of the transition in OI has
rather high energy, about 10.74 eV, relative to the ground state, hence, the strong
line intensity outside the core of the plasma (i.e., outside the white area of the con-
tour plot) indicates that plasma is in a non-equilibrium state with a strong presence
of high-energy electrons (1 eV) and an abundant concentration of 5P state OI in
352 S.P. Kuo et al.
the plasma effluent, again assuming no other emissions are present within the finite
filter pass-band. Since OI in other states (in particular, in the ground state) may also
be produced, the total OI concentration in the plasma effluent may be much higher
than that of 5P state alone.
The effectiveness of the plasma spray on stopping bleeding from a hole onto an ear
saphenous vein and a cut to an ear artery were examined. The natural clotting (bleed-
ing) times of the similar wounds on untreated controls were used as references for
comparisons.
Fig. 27.2 (a) A needle punching a hole in an ear saphenous vein, (b) blood flowing out of the hole,
(c) plasma treatment, and (d) bleeding stopped after 14 s (7 on/off runs) of plasma exposure
27 Non-equilibrium Air Plasma for Wound Bleeding Control 353
A saphenous vein from a pig ear was first identified; a needle was then used to
punch a hole in this vein as shown in Fig. 27.2a, in which the orange ring is an ID
tag. When blood flow started as shown in Fig. 27.2b, it was treated immediately by
the plasma spray with an exposure distance of 2.5 cm as shown in Fig. 27.2c. The
plasma spray was run with 2 s on and 2 s off alternately. After 7 runs, the bleeding
was stopped completely as demonstrated in Fig. 27.2d. The total exposure time to
the plasma spray was 14 s. In the untreated control, the bleeding time of the other
pig was measured to be about 88 s.
Before cutting an artery, the ear was tied with a tourniquet to slow down the blood
flow. A scalpel was then used to cut the ear small artery and then measure the natu-
ral coagulation time as well as the needed plasma treatment time. The processes of
the two cases, untreated and treated, are illustrated in Figs. 27.3a, and b, respec-
tively. The treatment was adopting an intermittent approach, plasma on-off alter-
nately with 2-s on 4-s off. It was found that it took 1 min to stop bleeding naturally.
On the other hand, bleeding was stopped after 6 runs of plasma on-off treatment.
Although the total treatment time was about 35 s, only about half of the natural clot-
ting time, the actual plasma treatment time was only 12 s.
Fig. 27.3 Photos showing the processes of cutting an artery, and (a) leaving the bleeding to stop
naturally and (b) stopping the bleeding by the plasma spray
354 S.P. Kuo et al.
Fig. 27.4 Progress of the recovery of treated (rows 1 and 2) and untreated (row 3) straight cuts
27 Non-equilibrium Air Plasma for Wound Bleeding Control 355
Fig. 27.5 Progress of the recovery of treated (rows 1 and 2) and untreated (row 3) cross cuts
In this study, we report in-vivo tests of blood coagulation assisted by a plasma spray.
The experimental results have demonstrated that this plasma spray could rapidly
stop bleeding. It was not easy to locate a saphenous vein in the body of the pig. Thus
an ear saphenous vein, which is a relatively small vein, was chosen in the test. With
this choice, it also reduced the blood loss of the pig used in the untreated control.
Yet, the plasma effluent reduced the bleeding time from 88 to 15 s. Since bleeding
from an artery is difficult to stop, again an ear artery was chosen for the test and the
ear was tied with a tourniquet before the cut. The results of the tests showed that the
plasma effluent can effectively clog the cut to the artery to stop bleeding; the bleed-
ing time was reduced to a half.
The experimental results have shown that this plasma spray could rapidly clot
blood to stop bleeding and made a positive impact on wound healing. The atomic
oxygen produced in the plasma effluent is likely the catalyst in the coagulation pro-
cesses. When interacted with H2O, atomic oxygen carried by the plasma effluent
can generate large amount of reactive oxygen species (oxygen ions, free radicals,
and peroxides). Studies have shown that platelets are a prime target for oxidants
produced or released in the vascular lumen and, at the same time, they are also
capable of endogenous generation of oxidants [18, 19]. It has also been shown that
oxidants can affect several key steps of platelet function to enhance platelet aggre-
gation [1921].
Hypoxia [22] acts a key factor to stimulus tissue repair by creating an oxygen
gradient from the hypoxic tissue of wound to the nearby unbroken tissue [23].The
central area of the wound is most hypoxic, and the oxygen gradient increase toward
the uninjured tissue progressively. However, with the supply of RAO from the
plasma spray, the amount of oxygen, consumed to generate H2O2, is reduced.
Consequently, more oxygen can be shared in other action such as producing super-
oxide (SOD), cell metabolism and raising tissue oxygen tension in the wound heal-
ing. RAO also provides the oxygen in the blood by reaction of catalase which plays
a protection role avoiding cells damaged by H2O2. In summary, RAO reduces the
demand of oxygen using in respiratory burst and increases oxygen content of tissue
356 S.P. Kuo et al.
indirectly. Both of reducing requirement and increasing supplement paths raise the
tissue oxygen tension in the wound site during the plasma treatment, as well as
provide the oxygen for wound healing and cell metabolisms
Acknowledgements We are grateful to Alessandro Betti for fabricating the plasma spray
device. This work was supported in part by a NYU-Poly seed Grant and in part by Adventix
Technologies Inc.
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27 Non-equilibrium Air Plasma for Wound Bleeding Control 357
Morphologies and functions of cells and their constituents can be changed by expo-
sures to electric fields. The response that is instigated depends on the strength of the
electric field and the duration it is acting upon the target. Short stimuli (on the order
of milliseconds and voltages of several tens of millivolts) that are imposed across
the cell membrane open voltage-gated channels and, in this way, regulate the trans-
port of ions such as potassium and sodium across the membrane [13]. Extended
exposures of several seconds or even minutes with several tens of volts per meter
provide enough energy to either denaturate proteins or even cause thermal damage
directly, i.e., burn tissues. Stimulations of the first kind are used in electrophysiolog-
ical studies of action potentials, for example. The second type of exposure, with the
goal of delivering energy to malignant tissues, is utilized in radio- or microwave
ablation therapies [4, 5]. Instead of these quasi-continuous exposures, temperatures
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 361
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_28, Springer Science+Business Media B.V. 2012
362 J.F. Kolb and M. Stacey
that are no longer conducive to survival can also be achieved with a single pulsed
electric field of short duration, if the field strength is sufficiently high. More inter-
esting, however, are the more specific responses that can be accomplished with
short, pulsed electric field exposures that will not result in thermal damage, but are
already considerably stronger than stimuli that would merely trigger a physiological
reaction by membrane proteins. Biological effects that are instigated above this
threshold are generally caused by an initial increase in the permeability of mem-
branes. This effect is generally attributed to the formation of pores. Characteristics
and dynamics, i.e., pore diameter, their distribution along the cell surface, and life-
time, are critically dependent on the pulse duration and magnitude of the applied
electric fields [610]. In addition, many applications take advantage of a cumulative
effect on the membrane and rely on exposures with bursts of individual pulsed elec-
tric fields. Biological processes, and ultimately, the fate of a cell, depend on the
possibility that these pores will reseal, and on the transport of substances across the
membrane, either by migration or diffusion, while they are open [1113].
Consequently, the delicate balance of ion concentrations of sodium, potassium,
chlorine, calcium and others will be disturbed. Moreover, large molecules, such as
pharmaceutical agents and genetic material, can pass this otherwise-impenetrable
barrier [14, 15]. After membrane integrity has been restored, these additions to the
cytoplasm will then participate in the cells biochemistry.
This offers intriguing possibilities for new medical therapies and novel biotech-
nological approaches. For example, can chemotherapeutic agents that are poorly
membrane permeant, and consequently require large doses to be effective, now be
efficiently delivered into tumor cells directly [14, 16]. Another appealing applica-
tion is the introduction of genetic material into cells to change their function and
development. The method is currently being investigated for its potential as a vac-
cination against certain cancers, while the most prominent use of the technique is
probably still in cloning [17, 18]. Of course, if exposure parameters prevent resto-
ration of membrane integrity, cells will die [13]. Accordingly, pulsed electric fields
have also been used successfully to ablate tissues and to inactivate microorgan-
isms, particularly in liquids where most current applications focus on the decon-
tamination of water and food, such as milk and juices [1921]. In fact, the killing
of bacteria and yeast in water by this method was already reported by Sale and
Hamilton in 1967, which is also commonly assumed to be the first account of
membrane damage by pulsed electric field [22, 23]. The process was later described
as electroporation by Neumann [24]. The name summarizes theories that explain
the damage to the membrane and lead to an increase in permeability by the forma-
tion of pores under the influence of the electric field. When exposure conditions
allow for membrane recovery, they may still have a temporary effect on microor-
ganisms without necessarily killing them. For aquatic species, e.g., brine shrimp, a
temporary inactivation or stunning is observed that might last several minutes
but does not seem to permanently impair the organisms functions [25]. Details of
the mechanisms responsible for the observed transient inactivation of organisms of
higher order are even less well studied and understood than for single cells.
However, an obvious application of the method is the prevention of biofouling in
28 Subcellular Biological Effects of Nanosecond Pulsed Electric Fields 363
water treatment facilities or the treatment of coolant water taken from reservoirs
(lakes and rivers) without the need for chemical solutions, and therefore, no envi-
ronmental burden [26].
Different applications and the ranges for pulse durations and pulse amplitudes nec-
essary to achieve a specific response not caused by thermal damage or by the phys-
iological activation of membrane proteins, are shown in Fig. 28.1. Most of these
applications assume that an increase in membrane permeability is the underlying
cause, and that this increase in permeability is primarily limited to the outer cell
membrane. Subsequent biological responses are a result of the different processes
that are set in motion by this damage. Whether pores form in the membrane depends
on the voltage difference that can be achieved across the membrane during the
exposure. Without damage, the cell membrane resembles a fairly good insulator.
Fig. 28.1 Parameter ranges for different applications of pulsed electric field exposures of cells.
A potential difference above a critical voltage, Vcr, will result in the formation of pores, which initi-
ate the subsequent response. For shorter pulse durations, increasingly higher voltages are required
to reach the critical voltage during exposure as indicated by the dash-dotted line. (The calculation
of exposure parameter for a given critical voltage, Vcr, is assuming a spherical cell of 10 mm,
suspended in a medium of 70 Wcm, equivalent to the conductivity of the cytoplasm.) The electrical
energy applied will also heat cells, and thermal damage becomes more prevalent with increasing
field strengths as indicated by the dashed line. (The calculation of the critical temperature increase,
DTcr, assumes an adiabatic heating of the cell volume filled with water)
364 J.F. Kolb and M. Stacey
Charges (sodium, potassium and other ions) accumulate along this barrier during
the application of an electric field, E, leading to a change in the transmembrane
potential, Vcell. When the membrane potential change reaches a critical value rang-
ing from several hundred millivolts to 1 V, pores will form. (The value varies
among cell types, but is close to 1 V for most cell membranes.) An extended expo-
sure primarily provides the energy to increase pore size and number [27].
For a spherical cell with diameter, D, the charging characteristic at the poles of
the cell (with respect to the direction of the field) is described by Eqs. 28.1 and 28.2
[28, 29]. (The angular dependency of the induced membrane potentials is described
by a cosine modulation of this maximum value not shown.) Hereby, the charging
time constant, tc, is again determined by the size of the cell, together with the spe-
cific membrane capacity, cm, and the conductivities of the cytosol, rc, and the cell
suspension, ra. Typical values for the resistivity of the cytoplasm for physiological
cell solutions are on the order of 70 Wcm, while values of 1 mF/cm2 have been deter-
mined for the specific membrane capacitance [30, 31].
D
| Vcell (t) | = 1.5 E (1 exp( t / c )) (28.1)
2
D
c = (c + 0.5 a ) c m (28.2)
2
d t t
| Vorganelle (t) | = (1.5)2 E exp( t / c ) c exp (28.3)
2 c o
c o
The charging time constant for the organelle, to, is determined by an expression
similar to Eq. 28.2, by replacing cell diameter, D, with organelle diameter, d, the
conductivity of the cytoplasm, rc, with the conductivity of the organelle content, ro,
and the ambient conductivity of the suspension, ra, with the conductivity of the cyto-
plasm, rc. (It is also assumed that the dielectric properties of all cellular membranes
are identical to the characteristics of the outer cell membrane.) The comparison with
the development of transmembrane voltages across the outer membrane shows that,
for sufficiently high electric fields, subcellular membranes will also experience
potential differences that are on the order of, or exceed, typical critical voltages. For
a 5-mm diameter organelle (e.g., the nucleus) inside a 10-mm cell, and an exposure to
a field of 10 kV/cm, the voltage changes after 5 ns across the membranes are
|DVorganelle| = 0.93 V and |DVcell| = 0.68 V, respectively. The example demonstrates that
subcellular membranes can in fact charge faster than the outer cell membrane.
Accordingly, poration of organelle membranes and modifications of subcellular struc-
tures can be expected for strong applied electric fields. The example further shows that
membranes are charged to critical voltages in only a few nanoseconds. Since the
extent of the exposure, after critical values are met, primarily determines the further
increase in pore density, short exposures on the order of, or shorter than, the charging
time of the outer cell membrane could have a more pronounced effect on organelles
than on the cell membrane. Moreover, even a less extensive poration of subcellular
membranes might be sufficient to trigger an irreversible biological response.
Equations 28.1, 28.2, and 28.3 give a simplified description of the mechanisms
following the application of an electric field. However, the approach cannot account
for many parameters of the exposure, such as actual subcellular geometries, mem-
brane compositions, or non-linear events, such as the change in membrane conduc-
tion after pores have formed. Even the application of a field with an infinitely fast rise
time is an idealization. More elaborate approaches have been employed to describe
actual experimental conditions more accurately and gain further insight. Most inter-
estingly, a more detailed analysis of the exposure to intense short pulsed electric
fields with a fast rise time predicts that, in fact, the pores that can be generated under
these conditions are different from pores that can be created by longer pulsed expo-
sures of lower field strength as commonly used in electroporation techniques for the
outer membrane. In particular, all membranes (outer cell membrane and organelle
membranes) are uniformly porated within a few tens of nanoseconds into the expo-
sure [7, 35, 39, 40]. The number and density of pores is predicted to be several orders
of magnitude higher when compared with electroporation pulses. However, pores are
also expected to be much smaller, which would allow only small ions to pass through
but not larger molecules [6, 41]. Since many of the ions that are involved in the regu-
lation of cellular functions still could permeate cellular membranes, cell functions
are likely to be affected. As a result, intense ultrashort pulsed electric field exposures
offer a method for intracellular manipulation of cells (Fig. 28.1).
366 J.F. Kolb and M. Stacey
The theoretical analysis shows that exposures that primarily affect subcellular
structures require pulsed electric fields with a duration that is short compared to the
charging time of the cell envelope. (For a cell of 10 mm in diameter and resistivities
for cell suspension and cytoplasm of 70 Wcm, the charging time constant is 52.5 ns
and the charging time is, accordingly, on the order of 200 ns.) In addition, the field
strengths need to rise as fast as possible to amplitudes in the range of several tens of
kilovolts per centimeter to expose also the subcellular space before it is shielded and
critical voltages can no longer be achieved in the remaining field. These parameters
cannot be provided using conventional electronic circuits at least not for the expo-
sure of relevant cell or tissue volumes between electrodes at least a few millimeters
apart. Alternatively, pulsed power technologies are employed [42]. Basic circuits
are based on pulse forming networks or pulse forming lines. When using a pressur-
ized spark gap as the switching element, rise times of 1 ns have been realized for
voltage pulses with amplitudes of 35 kV, corresponding to electric fields of 350 kV/
cm across the electrode gap of 1 mm of a standard electroporation-cuvette [43].
The equipment allows the exposure of 100 ml of cell suspensions, which is sufficient
for the post-exposure evaluation of biological and biochemical responses in particu-
lar. (For lower electric fields, larger volumes can be exposed.) Similar systems have
also been used in in vivo experiments. In this case, high voltage pulses were usually
delivered by needle electrodes instead of plane parallel electrodes [4446]. For the
observation of early processes during or immediately after the exposure, micro-
scope-mounted systems have been developed [4749].
The fastest mechanism that has been observed so far after the exposure to nano-
second pulsed electric fields is the charging of the outer cell membrane [47]. A fast
voltage sensitive fluorescent dye was used, reflecting changes in the electric field
across the membrane by characteristic changes in excitation and emission spectra.
These changes were recorded with a temporal resolution of 5 ns and quantified to
describe the development of the associated transmembrane potential. An example
of the measurements is shown in Fig. 28.2.
The highest change in voltage (about 1.6 V) was observed at the anodic pole of
the cell (the side facing the anode in a parallel plate exposure system). The change
corresponds approximately to the values expected from theoretical models [50].
However, a significant difference has been observed between the anode and cathode
poles, with voltages that are 1 V lower at the cathode pole. Transmembrane voltages
across the cathode side also develop more slowly than for the anode side, which
suggests significant contributions to the potential difference across the membrane
from dipole alignment. These dipoles are found in headgroups of phospholipids that
are embedded in the membrane. (In addition dipole moments might also be induced
by the electric field.) The restricted mobility of the molecules might account for the
observed differences between hemispheres [5153]. The time at which the peak
value in transmembrane potential is approached during the exposure depends on the
28 Subcellular Biological Effects of Nanosecond Pulsed Electric Fields 367
Fig. 28.2 Transmembrane voltage changes (absolute values) of Jurkat cells for the exposure to a
50-kV/cm electric field of 60-ns duration (indicated by the shaded area). Values at the anode pole
jump to more than 1 V immediately. The difference in the values across anode and cathode might
be accounted for by the alignment of phospholipid heads, which then affect the local electric field.
Pores gradually open across the membrane during exposure and, after reaching a peak value of
about 1.4 V, allow for significant discharge currents
amplitude of the applied electric field. After the peak voltage is reached, values start
to decrease again. Apparently pores are starting to open across the membrane very
early during the exposure. Their continuous increase in number would allow for a
limited migration of ions, which first impedes charging and eventually will lead to
a reversal and discharge the membranes through these leaks at an increasing
rate. That the observed charging mechanisms are in fact primarily determined by
physical parameters was recently confirmed by similar experiments conducted in
plant cells [54].
Due to the significance of the cell membrane as the interface for the cell to receive
and process outside stimuli, and to the readily available methods to study these
interactions, many studies have focused on the effects on the outer membrane [5561].
They have confirmed that the membrane initially becomes permeable for smaller
ions only. However, for relatively long exposures and relatively high electric fields,
it is possible that the membrane eventually becomes permeable for larger molecules
[60, 61]. Some experiments on the permeability for small ions show certain other
characteristics, such as ion selectivity or preferences of ion movements that cannot
be explained by simple diffusion through holes in the cell envelope alone [58, 62].
Likewise this model cannot sufficiently explain the observed translocation of phos-
phatidylserine to the outer cell surface [63, 64]. (The membrane protein is usually
found exclusively on the inside of healthy cells and expressed on the outside only
when cells undergo apoptosis.)
368 J.F. Kolb and M. Stacey
Fig. 28.3 The cytoskeleton of HeLa cells was made visible by staining actin filaments (with
Oregon green 488 phalloidin). The cells were growing attached to polylysine-coated coverslips.
Image (a) shows the extensive and intact cytoskeleton typical of control cells (sham exposed).
Image (b) shows cells 1 min after exposure to a single 60-ns pulsed electric field of 60 kV/cm. The
membrane appears ruffled and filaments become less distinct. Four minutes after exposure, the
cytoskeleton structure disappears and bright actin spots appear instead. Simultaneously, the cell
shape becomes spherical and cells detach from the cover slip
DNA extracted immediately after exposure. Direct effects on the nucleus have also
been found with DNA markers, such as acridine orange [61]. The dye is used as a
nuclear stain, intercalating with the double strand structure of the DNA. Immediately
after the exposure to a nanosecond pulsed electric field, the recorded fluorescence
activity decreases. This observation can be interpreted either as a direct effect on the
binding sites between DNA molecules and dye, or as an outflow of dye through
pores that are forming in the nuclear envelope.
Notwithstanding the significant direct effect on macromolecules and struc-
tures, the exchange of molecules and ions across barriers through pores formed by
the exposure is still likely to be the most important mechanism to affect cell func-
tions. The concentrations of many of the ions released into the cytosol this way
are, under ordinary circumstances, carefully maintained. A sudden change in con-
centration will lead to a response with the goal of compensating for the imbal-
ance. Since changes in ion concentrations regulate cell functions, nanosecond
pulsed electric fields should likewise provide control of these mechanisms. First
proof of this concept was provided in an experiment conducted by Stephen
Buescher with neutrophil chemotaxis. When placed in a microreactor between two
electrodes, the cells move towards a chemoattractant. The directed movement
is temporarily interrupted by the application of a single 300 ns, 45 kV/cm
pulse [73].
One of the most important ions involved in intracellular signaling events and
chemical signal transmission between cells is calcium. It is stored inside cells in
the endoplasmic reticulum and mitochondria and is released in small quantities
for signaling events. Cell functions that are mediated by varying calcium concen-
trations include fertilization, muscle contraction and apoptosis. Calcium responses
370 J.F. Kolb and M. Stacey
Fig. 28.4 A single pulsed electric field of 60 ns instigates an immediate, transient release of cal-
cium from intracellular stores (as indicated by the fluorophore fluo-4). The magnitude of the fluo-
rescence response increases with field strength. For fields of 50 kV/cm, the induced signals are of
the same order of magnitude as subsequent naturally occurring fluctuations. However, for a field of
100 kV/cm, these random changes no longer occur, indicating sustained subcellular damage. This
seems to be confirmed by the instantaneous drop in fluorescence after several minutes, suggesting
loss of membrane integrity
induced with nanosecond pulsed electric fields have been observed in a variety of
cell types with different pulse parameters [7376]. A detailed analysis shows that
the release of calcium from internal stores occurs within only a few milliseconds
after exposure, again indicating that pores, rather than physiological pathways,
are responsible [77]. The calcium response is transient and qualitatively similar to
physiological signals, as observed for a normal, i.e., unperturbed, cell. This shows
that cells deal with the electrical stimulus in a manner similar to other trigger
mechanisms, and calcium is eventually returned to these intracellular stores
through calcium pumps. How much calcium is released depends on the pulse
parameters (Fig. 28.4). When only moderate increases in concentrations are insti-
gated, calcium activated channels do not seem to activate in the outer cell mem-
brane, and no calcium is taken up from outside. However, if the pulse amplitude
is large (with respect to pulse duration and cell type), calcium pathways can incur
significant damage. Calcium rushes in through the cell membrane, further increas-
ing calcium concentrations. In this case, cells wont recover from the stimulus; in
fact, after several minutes, loss of membrane integrity can be observed, which
indicates the death of the cell.
28 Subcellular Biological Effects of Nanosecond Pulsed Electric Fields 371
As a tool, pulsed electric field exposures offer interesting possibilities for the treat-
ment of cells in medical and environmental applications. The potential to disrupt
cell membranes and subcellular components with the goal of killing cells is appar-
ent [19]. As such, the method is particularly interesting and is investigated to treat
contamination of liquid foods, e.g. milk and fruit juices, and also for the treatment
of waste water and drinking water [20, 25, 8085]. For nanosecond pulsed electric
fields, the stimulus can actually reach inside the cell and be used against pathogens
that are otherwise protected against agents acting on the cell membrane or chemi-
cals that need to permeate the membrane first [86, 87]. The lack of chemical residue
is a further advantage for environmental applications.
The unique strength of the method, however, lies in the possibility of instigating
more subtle responses [67, 72, 88, 89]. Sub-lethal exposure conditions still hold the
potential to affect internal structures and membranes. Many of the direct mecha-
nisms are speculative but it seems plausible that proteins, e.g., receptors, can be
directly affected, for example, by breaking individual molecular bonds or by induc-
ing charge shifts along macromolecules. Subsequently, cells will respond with a
characteristic signaling cascade and coping mechanism if the stimulus can mimic a
familiar stimulus, for example, from a chemical compound. Alternatively, protein
structures might actually be broken and cells will have to expend repair mecha-
nisms, possibly leading to unforeseen results in the attempt to repair or compensate
for the damage [90, 91]. Even the relatively simple process of releasing ions from
internal stores, in particular calcium [73, 74, 77], will first of all be interpreted as a
biochemical signal. Many of the cell functions that are controlled by intracellular
calcium concentrations will respond accordingly [73]. This offers a particular means
to control the behavior of specialized cells. These include excitable cells, and
accordingly, the effect of nanosecond pulsed electric fields on cells have been shown
for cardiac myocytes [92], skeletal muscle [93], motoneurons [94], and neurosecre-
tory cells [95].
When applied to platelets, pulsed electric field exposures have been shown to
instigate the same response as the enzyme thrombin. The process is also mediated
by an increase in intracellular calcium concentrations. As a result, platelets aggre-
gate [96]. (Platelets are specialized blood cells that are activated to aggregate in
wounds and contribute to coagulation.) Platelet rich plasma is used in surgical and
chronic wound care. The activation using the physical (electrical) stimulus avoids
complications that have been associated with the use of bovine thrombin and further
372 J.F. Kolb and M. Stacey
eliminate the dependency on the protein. Studies that have been conducted to deter-
mine the healing rate using platelet gels activated by pulsed electric fields show that
wounds heal at least as fast as when the gels are activated with thrombin. In addi-
tion, a bactericidal effect of the electric field activated gel was observed, which
would help to prevent infections.
Perhaps the most interesting application of nanosecond pulsed electric fields
involves their capacity to induce apoptosis in cancer cells. (Apoptosis is also known
as programmed cell death a process that is inhibited in cancer cells, leading to
uncontrolled cell proliferation.) The interaction mechanisms are still under investi-
gation, but in general, many different processes could be affected by exposure to an
electric field [41, 97]. Accordingly, different pathways have been investigated [57,
60, 61, 63, 67, 78, 98]. Apoptosis can be instigated by the effect on either the outer
membrane or on organelles, such as the mitochondria. Hallmarks of apoptosis, such
as activation of caspases, release of cytochrome c, phosphatidylserine externaliza-
tion and DNA fragmentation, have been observed and studied with respect to expo-
sure conditions [46, 78, 79, 99102].
More recently, the efficacy of pulsed electric fields alone as a tumor therapy has
been tested in in vivo experiments on different tumor types. Figure 28.5 shows first
results for B16 melanoma tumors grown in mouse skin. By applying 100 pulses of
300 ns duration and 10 kV amplitude in different locations across the tumor, a
significant reduction in tumor size could be achieved in only one day. No chemo-
therapeutic drugs were administered to enhance this effect. In subsequent studies,
the treatment conditions were optimized and eventually a complete remission of
tumors was achieved in a group of 17 animals with a treatment regimen applying
up to 100 pulses of 300-ns, on different days during a 2 week period [103]. (The
number of treatments depended on the individual tumor response.) While more of
half of the 18 control animals did not survive for more than 3 weeks (and few did
much beyond that), did all of the treated animals live for more than 120 days, the
commonly accepted point demonstrating long term survival. Tumor cells treated
in vivo showed many of the same hallmarks of apoptosis that were already observed
for cell suspensions and some other features, such as characteristic nuclear shrink-
age [44, 45, 104]. In addition, nanosecond pulsed electric fields showed the ability
to act not only on individual cells, but also systemically on the tumor by disrupting
the capability for angiogenesis, hence disrupting the tumors supply of nutrients
from blood [44, 45]. In the meantime, similar results and long term survival could
also be demonstrated on HEP1-6 liver tumors that were also grown subcutaneously
in a mouse model and treated with 100-ns pulses. Many other tumor cell lines have
been investigated for their susceptibility, at least in vitro, but some additional ones,
in vivo [46, 71, 79, 89]. In one case, a basal cell carcinoma was successfully treated
in a patient [46].
Although nanosecond pulsed electric field treatments do not require additional
agents, such as chemotherapeutics, to be effective, possible synergies might actually
enhance a sought-after effect, for example, by increasing the permeability of subcel-
lular membranes for chemical compounds [65, 105]. The possibility to reach into
the cell with an electric field could also offer a way to control cell functions remotely
28 Subcellular Biological Effects of Nanosecond Pulsed Electric Fields 373
Fig. 28.5 Results of a pilot study comparing untreated melanoma tumors (right column) and mel-
anoma tumors that were treated with a nanosecond pulsed electric field regimen (left column). One
million B16f murine melanoma cells were injected into the flanks of C57BL6 mice. When tumors
reached a diameter of about 5 mm, nanosecond pulsed electric fields were applied by inserting a
pair of needle electrodes (31 gauge hypodermic needles) on either side of the tumor. The electric
field was generated by applying a 10-kV pulse of 300-ns duration (30 ns rise time) from a Blumlein
pulse forming network. For the best possible exposure, the electrodes were relocated in 34 steps
(depending on tumor size) of 1 mm and the treatment repeated at the new location. In each loca-
tion, 100 pulses were applied. The upper left picture shows the tumor immediately after the first
treatment with injection sites clearly visible. The treatment was repeated on the next day. The
lower left picture shows the same tumor on the third day. The treated tumor is regressing rapidly,
while the control tumor almost doubles in size over the same period
by introducing otherwise inactive substances. A first attempt has been made using
carbon nanotubes, which were introduced into tumor cells and are expected to
respond strongly to an applied electric field due to their unique electrical character-
istics [106]. Even these newer developments, however, rely on electrode systems
that can be brought close to the tumors. Accordingly, targets that can be treated have
to be located close to the skin surface or they will require invasive surgery. A new
idea proposes to focus strong electric fields into a patient by using ultrawideband
antennas [107]. This appealing approach requires shortening the high voltage pulses
that have to be applied to the antenna into the picosecond range and increase the
amplitudes to several tens to hundreds of megavolts per centimeter. Accessing this
parameter range poses new challenges and opportunities for engineering and
research. Different physical phenomena and processes will be dominant for these
conditions, and as a consequence, will likely lead to different biological responses
as it has already been observed for cell viability [108].
374 J.F. Kolb and M. Stacey
Acknowledgment The insight and knowledge on effects and applications of pulsed electric
fields is the result of a dedicated group of researchers on this topic. I have been lucky to work
with many of them at the Frank Reidy Research Center for Bioelectrics or to have found them as
collaborators at Old Dominion University. Without their ideas and dedication, the field would
not have advanced as it has. Credit and my thanks for the opportunity to write this summary
therefore are extended to E. Stephen Buescher, Peter F. Blackmore, Michael Stacey, Stephen J.
Beebe, James R. Swanson, Christopher Osgood, Ravindra P. Joshi, Shu Xiao, M. Arif Malik,
Yeong-Jer Chen, Richard Heller, Loree Heller, Olga Pakhomova, Andrei Pakhomov, Barbara
Hargrave, Richard Nuccitelli, Angela M. Bowman, Betsy Gregory, W. Hunter Baldwin, Jennifer
Pomicter, Wolfgang Frey, Uwe Pliquett, Jue Zhang, Barbara Carroll (also for proofreading this
and many other manuscripts), Ruth Lyman, many students (too many to list them all) who spent
years on the actual experiments and in particular Karl H. Schoenbach for his vision and lead-
ership in this field.
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intense subnanosecond electrical pulses on biological cells. IEEE Trans Plasma Sci
36:414422
Chapter 29
First Achievements and Opportunities
for Cancer Treatment Using
Non-thermal Plasma
Abstract This paper summarizes the experimental results and plasma delivery
strategy developed in Orlans for the evaluation of antitumor action of dielectric
barrier discharge and plasma gun for cancer treatment. Detailed analysis of biologi-
cal effects following non thermal plasma application for both in vitro and in vivo
experiments reveals the role of ROS, DNA damage induction, cell cycle modifica-
tion and apoptosis induction. Recent characterization of plasma splitting and mixing
in different capillary geometries, using the plasma gun, together with preliminary
tolerance study dealing with lung and colon treatment indicate that endoscopic
plasma delivery may be a new and valuable therapy in cancerology.
29.1 Introduction
This work deals with the development of two non thermal plasma (NTP) sources
and their application in cancerology. A floating electrode dielectric barrier discharge
device (FE-DBD), close to that first proposed by Drexel Plasma Institute [1], and a
plasma jet generator, labelled plasma gun [2], are used for both in vitro and in vivo
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 381
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_29, Springer Science+Business Media B.V. 2012
382 E. Robert et al.
assessment of non thermal atmospheric pressure plasma as a new therapy for cancer.
Antitumor activity of NTP was evaluated in vitro on U87 glioma cancer and HCT116
colorectal cancer cell lines. Cell cycle analysis, DNA damage characterization,
reactive oxygen species (ROS) quenching, and apoptosis quantification were per-
formed to allow the description of the processes involved in the plasma antitumor
action. Antitumor effect of plasma on in vivo U87 xenografs subcutaneously
implanted on mice legs was previously reported [3]. In this work, complementary
biological diagnostics have been used to allow, for the first time to our knowledge,
in vivo apoptosis and cell cycle alteration measurements after NTP treatment. The
agreement between in vitro and in vivo NTP induced effects are enlighten and sup-
port the development of the Plasma Gun for its optimization in plasma endoscopy.
Preliminary encouraging NTP endoscopy feasibility studies and tolerance assess-
ment are finally reported. In a first section, the two NTP sources developed in
GREMI are briefly presented. The Sect. 29. 2 summarizes the in vivo and in vitro
antitumor studies together with a first approach of possible process chain to explain
in vitro and in vivo antitumor action of NTP. The specific features of the Plasma
Gun associated with the propagation in thin, long and eventually branched capillar-
ies are documented in Sect. 29.3 before some concluding remarks.
Figure 29.1 presents a synopsis and a picture of each of the two plasma sources
developed in GREMI. While being cheap, robust and rather easy to develop, the
FE-DBD source suffers from several limitations, inherent with its mode of opera-
tion, which do not preclude its use for NTP assessment but support the design of
new NTP source much more convenient and matched for in vivo applications. The
generation of NTP with FE-DBD devices, occurs when the gap between the dielec-
tric embedded powered electrode surface and the sample ranges from about 1 to
5 mm. The sample (cell culture dish or living tissue) acts as a floating potential
electrode through which the low amplitude (mA range) electrical current circulates.
When the gap thickness is larger than about 5 mm, the plasma generation stops for
the typical voltage, ranging from about 10 to 50 kV, used to sustain the discharge
through millimetric dielectric barrier layers. The safe use of FE-DBD for in vivo
applications was clearly demonstrated, together with the possibility to induce very
limited undesirable effects such as skin burns, local temperature increase, etc., if the
so called plasma dose is limited to a few J/cm2 [4]. Nevertheless, the application
of high voltage pulses, a few tens of kV in amplitude, across a dielectric barrier in
the very close vicinity of the biological target may require some specific safety
precaution to anticipate the possible dielectric layer disruption and the successive
arc burning from the powered electrode to the sample. The possibility to develop a
small volume DBD applicator, likely to be used for endoscopic treatment also seems
rather complex even if DBD plasma were recently designed through flexible capil-
lary [5]. In this latter case, the production of DBD plasma all along the capillary
29 First Achievements and Opportunities for Cancer Treatment 383
Fig. 29.1 Left: synopsis and photography of the FE-DBD source, the discharge operates in air.
Right: synopsis and photography of the Plasma Gun source, the discharge operates in neon
the biological sample. The plasma delivery can thus occur at large distances, a few
tens of centimetres, away from the high voltage reactor, through flexible capillary of
various nature and diameters down to a few hundreds of microns. The plasma at the
capillary outlet may extend up to a few centimetres downstream where energy trans-
fers occur leading to a plasma composition changing. An important difference
between the two NTP sources lies in the decoupling of the plasma generation and
transport, with the plasma tailoring at the target surface.
The first experiments were performed using the FE-DBD as an external plasma
applicator. For in vitro studies, two different cell lines U87 (human glyoblastome)
and HCT116 (colon cancer) were cultured and transferred into 24 well plates con-
taining 500 ml of culture medium. The gap between the DBD reactor and the upper
surface of the culture medium was set to 2 mm. The DBD was applied during a
preset period of few seconds at a repetition rate of 2 kHz, the voltage pulse ampli-
tude being of 25 kV, the pulse duration of 5 ms (fwhm).
Both cell lines are transfected to express firefly luciferase, an enzyme catalyzing
light production thanks to luciferine substrate, allowing in vitro and in vivo biolu-
minescence imaging. This imaging modality appears as a unique tool, complemen-
tary with the calliper measurement, as bioluminescence signal intensity is related
with the tumor metabolism rather than with the tumor volume which may include
necrotic areas. Bioluminescence signal is produced by alived cells, its intensity is
correlated with the tumor metabolism. The volume is inferred from the measure-
ment with the calliper of two perpendicular diameters of the tumor following the
international standard technique. In vivo experiments were performed using nude
mice, following the international animal care guidelines (EC directive 86/609/CEE,
French decree n 87848). Tumor xenografs were achieved by subcutaneous injec-
tion of tumor cell suspensions (106 cells in 0.1 ml 0.9% NaCl) into the hind legs.
The operation of the FE-DBD at low electrical power, and correspondingly mod-
erate plasma flux on the mouse skin surface, allows repetitive plasma delivering up
to 6 min during 5 consecutive days. Following this plasma application protocol,
neither systemic behaviour, cardiac and pulmonary rhythm alterations nor severe
skin burns were measured during tolerance studies [3].
A five day DBD plasma treatment, with a daily 6 min at 200 Hz plasma fraction,
was shown to induce a significant delay and growth rate reduction of U87 glioma
cancer, in comparison with non treated control group [3]. Both short term and long
term effects were observed and demonstrate, for the first time to our knowledge, the
NTP potentialities for in vivo cancer treatment approach. Following this encourag-
ing in vivo trial, a detailed analysis of plasma induced effects on in vitro cancer cells
was performed. As verified by different authors, the indirect treatment for which the
culture medium is exposed to plasma treatment before being transferred to wells
containing cells, was shown to induce a comparable cell death with that resulting
29 First Achievements and Opportunities for Cancer Treatment 385
Fig. 29.2 U87 cell cycle distribution in control and plasma treated groups for in vitro (left) and
in vivo (right)
form the direct plasma protocol. This is an indication that active species, such as
ROS, are the main agent in the plasma responsible for cell death for in vivo applica-
tions. This was confirmed by using a ROS scavenger in the culture medium before
plasma treatment. In this case, the plasma action is quenched, the bioluminescence
signal being comparable with that of the control wells. As illustrated in Fig. 29.2a,
the plasma application leads to significant cell cycle distribution modification. With
respect to the control groups, the flow cytometry measurement reveals that cells
exposed to plasma present a slight decrease on the population in the primary G0/G1
phase, an increase of the cell number in the S phase and no statistical variation of
the distribution in the G2/M phase. Same analysis was performed on the tumor
xenografts treated by DBD. Following the 5 day treatment, tumor fragments were
collected and analyzed by flow cytometry. Figure 29.2b presents this in vivo cell
cycle distribution measurement for treated and control mice. This figure indicates
that cell cycle distribution pattern is modified following in vivo plasma treatment
and that there exists a good correlation between in vitro and in vivo results.
The in vitro observation of cell death amplification 24 h and 48 h after the very
short duration (a few seconds) plasma treatment, and the in vivo long term effect
measurements suggested that plasma action relies essentially in cell death triggering
rather than in a physical instantaneous damaging. Figure 29.3 presents the biolumi-
nescence signal measured in control and plasma treated groups 6, 24, 48 and 72 h
after treatment completion. In the control group, the cell activity is continuously
growing with a very important increase from day to day up to the third day when the
cells have colonized the whole dish. The plasma-treated cell bioluminescence signal
is slightly increased 24 h after treatment and vanishes later. This programmed cell
death induction, so called apoptosis, was already proven to be of major importance
in NTP applications. In this work, in vitro apoptosis was measured 24 h after plasma
treatment using annexin V detection kit while in vivo apoptosis was quantified by
immunohistochemistry detection of cleaved caspase 3. For in vivo apoptosis
diagnostics, both control and plasma exposed tumors were excised 24 h after the
end of the treatment and tissue slices were collected with reference to the plasma
application position.
386 E. Robert et al.
1.01008
30s
6.01007
4.01007
2.01007
0.0
6h 24h 48h 72h 6h 24h 48h 72h
Fig. 29.3 Kinetics of bioluminescence intensities for control and plasma treated HCT116 cell
lines. The DBD plasma was applied for 30 s
Figure 29.4 presents the apoptosis index in control and plasma treated groups for
in vitro and in vivo U87 cell lines. In both cases, significant apoptosis induction is
measured 24 h after plasma treatment. As underlined from cell cycle analysis, the
apoptosis enhancement is of the same magnitude for in vitro and in vivo samples
exposed to different number of DBD plasma impulsions. As an illustration, a three-
fold apoptosis induction is measured whether in vivo after application of 3.6105
plasma pulses or in vitro as the result of 3104 plasma pulse delivery. These num-
bers simply indicate that comparable apoptosis induction or cell cycle modification
can be measured as a result of DBD application during a few seconds for cultured
cells, or during a few minutes over the skin surface. The picture in Fig. 29.4c pres-
ents the apoptotic cell distribution in a tumor slice. It has been measured that apop-
totic cells were homogeneously distributed among the whole tumor volume. While
the processes at the origin of plasma-induced in vivo modifications have not yet
been determined, our study indicates that they can be induced through rather thick
tissue layers and that no diffusion-like processes seem to be clearly evidenced.
In vitro DNA damages were analyzed by assessment of g H2AX immunofluores-
cence by flow cytometry. A very early response to DNA damage consists in the
phosphorylation of H2AX histone, leading to the recruitment of the gH2AX protein
at DNA damaged sites. The phosphorylation of H2AX can be detected with a spe-
cific targeted antibody. The fluorescence of such antibody tagged with specific fluo-
rescent marquer is a very sensitive diagnostics tool. In this work, assessment of
gH2AX immunofluorescence (IF) by flow cytometry was performed 1 h after treat-
ment using gH2AX phosphorylation Assay Kit (Millipore) in accordance with man-
29 First Achievements and Opportunities for Cancer Treatment 387
a 25 b 20
**
Apoptototic Cells (%)
5
5
0 0
CTRL 30s CTRL NTP
c
CONTROL PLASMA TREATED
Fig. 29.4 (a) in vitro apoptosis quantification after 30 s DBD treatment. (b) In vivo apoptosis
measurement, and (c) micropictures of caspase 3 fluorescence (red dots for on-line version) in U87
tumor. Higher apoptotic cell density zones appear as dark areas on the black and white picture
ufacturer protocol. Briefly, after ethanol fixation and saponin cell permeabilization,
histone H2AX phosphorylated at serine 139 was detected through the fluorescence
of the anti-phospho-Histone gH2AX, FITC conjugate.
Figure 29.5 presents the immunofluorescence signal intensity measured in U87
control and plasma treated cell lines. NTP treatment is shown to induce a significant
40% increase of the immunofluorescence level following 30 s of DBD application.
Such effects were detected as soon as 1 h after plasma treatment, suggesting that the
major contribution to DNA strand break is a direct consequence of plasma treatment,
388 E. Robert et al.
while DNA fragmentation associated with apoptosis induction would require longer
delays after treatment to be triggered.
As a summary, in vivo and in vitro analysis of plasma-induced antitumor effects
present striking common features. To the best of our knowledge, our study is the first
to demonstrate in vivo antitumor action of NTP together with the quantification of
cell cycle alteration and apoptosis induction. The major ROS implication for in vitro
experiments was clearly demonstrated and the early stage DNA damage measure-
ments allow to suggest a full plasma action scheme very close to that at the base of
the todays cancer treatment strategies, chemo and radio therapies. ROS chemo deliv-
ery or ROS generation through ionizing radiation targeting, induce lethal DNA dam-
ages leading successively to cell cycle modification and apoptosis triggering. It
appears that same chain processes occur following NTP application. The role of ROS
during in vivo treatments remains unclear, the main issue lying in the possibility for
these active species to pass through the mouse skin and diffuse to and inside the
tumor volume. Another scenario relies on the plasma-triggered, in situ, i.e. in the
tumor environment, ROS release which may be mediated by local charge density, pH
modification over the mouse skin under treatment. Such local pH modification was
for instance observed on the skin surface where acidification occurs while subcutane-
ously a slight increase of the pH value was measured. Such extracellular pH variation
may for instance influence the NO or hydroxyl radical production, and induce suc-
cessive DNA synthesis perturbation or apoptosis [8]. Besides the need for specific
study to determine whether in situ plasma triggered ROS release is a relevant assump-
tion to explain in vivo NTP mode of action, if ROS are the key agent for tumor cell
destruction, the tumor treatment will probably be greatly optimized by delivering
chemically active plasma in the close vicinity of the targeted cells. Tolerance studies
using FE-DBD on mouse colon were performed and demonstrate the possibility to
apply DBD plasma during periods of a few minutes. This is a proof that NTP could
be delivered even on rather fragile organs. The main targets of our study concern
colorectal and lung cancers for which organ access requires specific care and should
rather be operated through endoscopic treatment. The plasma gun, exposed in the
first section of this manuscript, may represent a unique device to evaluate the NTP
antitumor effect for these two challenging cancers.
The plasma gun developed for cancer treatment consist in a compact, see Fig. 29.1, low
power, a few to a few tens of watt, pulse power generator sustaining a nanosecond DBD
developing across glass pipe flushed with rare gases and rare gas based mixtures. The
outlet of the glass pipe is connected to thin flexible silicon or polyimide tubes through
which fast travelling, so called plasma bullets [9] propagate at very high velocity
towards the biological target. Besides the determination of the physical processes at the
origin of plasma propagation at very high velocities, up to a few 108 cm s1, recent
experiments have been performed to study the plasma expansion through micro sized
29 First Achievements and Opportunities for Cancer Treatment 389
Fig. 29.6 Five nanoseconds snapshots revealing plasma stream splitting in a T-shaped glass 4 mm
inner diameter. The plasma is generated in neon at atmospheric pressure. Time origin is set to 0 for
the first (left) picture, delay with respect to this origin are of 60, 80, 100, 120 and 140 ns from
second picture to the righter picture
in diameter silicon capillaries which can be used for mice tracheal intubation or colon
endoscopy. The plasma delivery can either be planed as a direct exposure of the tumor
surface or for less accessible targets through the plasma propagation in the organ
cavities leading to the cancerous location. The first technique requires implementing
non intrusive diagnostic to monitor the capillary outlet position while the second pro-
tocol imposes preliminary experiments on the possibility for plasma splitting, expan-
sion, shrinking, and connection as may be encountered in real organ topography. To
this end, the plasma splitting and mixing have been studied in multi branched glass
assemblies and in circular rings, using fast ICCD imaging. Both plasma splitting in
glass assembly presenting up to ten successive branchings, and mixing of two collid-
ing plasmas have been observed and characterized [10, 11]. The denomination of
Pulsed Atmospheric Plasma Streams (PAPS) was suggested to describe both the highly
emissive head of PAPS, so called bullet, and the very fast travelling filamentary
plasma region, appearing as a tail of the bullet, during the PAPS propagation [11].
Figure 29.6 presents, 5 ns duration, ICCD images showing the propagation and split-
ting of plasma streams in a T shaped glass expansion volume. These measurements
demonstrate the possibility to launch a plasma stream from a primary pipe and achieve
plasma propagation in successive bifurcations of a complex volume.
Plasma propagation in 200 mm inner diameter pipette was also characterized
together with the splitting of an initial plasma stream in three sister PAPS in a
cross shaped 200 mm inner diameter assembly as documented in Fig. 29.7. These
encouraging properties of atmospheric pressure plasma gun device, lead us to per-
form preliminary endoscopic experiment on mice both for lung and colon targets.
The propagation of plasma through small diameter flexible capillaries flushed at
moderate gas flow rate, 1050 cc/min, in the lung and colon of anesthetized mice
was recently successfully achieved together with preliminary evidence for a good
tolerance of both colorectal and lung tissues under plasma exposure up to 10 min.
The feasibility of such plasma application in combination with a proper matching of
the plasma gun characteristics allows in situ in vivo study and optimization of anti-
tumor action of NTP. Recent studies from other groups on the plasma jet action [12]
on colorectal cell lines confirms cell growth arrest [13] and report a selective apop-
tosis induction for different tumoral and normal cells [14].
390 E. Robert et al.
Fig. 29.7 Ten nanoseconds snapshot of plasma stream splitting in a 200 mm inner diameter cross
shaped glass assembly. The dark central region consists in an opaque mechanical connection of
four glass pipettes. Plasma is launched in atmospheric pressure neon gas in the pipe on the left
hand side of the photography
29.5 Conclusion
In vivo and in vitro NTP action have been tested and characterized on cancerous cell
lines. This work summarizes the detailed analysis of in vitro cancer cell lines, U87
and HCT116, response to non thermal plasma treatment. For this study, a floating
electrode dielectric barrier discharge set up, delivering microsecond pulses, was used
as a plasma applicator in ambient air 2 mm above culture medium surface.
Complementary analysis of, previously reported [3] antitumor action of the same
plasma source on U87 tumor xenografts implanted on mice groups, have also been
performed. In vitro plasma action appears to be largely induced by ROS production
in the culture medium, which then triggers a rather conventional cascade leading
to the cell destruction. Thus, early DNA damages have been detected and quantified,
successive cell cycle alteration leading to an accumulation of cell in a premitotic
stage, and significant apoptosis induction were proven for both cell lines. Cell cycle
modification and apoptosis induction were also proven to occur following in vivo
U87 plasma treatment. These two plasma-induced biological effects probably associ-
ated with the measured tumor short term activity stabilization and longer term growth
slowing, exhibit striking similarities both in vitro and in vivo samples. This would
suggest a major implication of ROS for in vivo NTP action. Two mechanisms appear
likely to be at the origin of ROS stimulation in the tumor environment. Whether ROS
produced in the air plasma diffuse through the mouse skin and tissue towards the
tumor vicinity, or plasma acts as an external trigger for ROS release in the living
organism. The first process may be favorized by synergetic action of high amplitude
transient electric fields generated in the DBD streamers and ROS penetration.
29 First Achievements and Opportunities for Cancer Treatment 391
The second assumption which is already known for instance in drug delivery for
melanoma treatment, or for arterial pressure control through NO release at selective
dosing, may be involved during NTP treatment. pH modifications or surface charg-
ing occurring during DBD plasma application, may be at the origin of such intra
organism ROS release triggering. Finally, the development of a specific plasma jet,
labeled Plasma Gun, is documented in two ways. First, atmospheric pressure plasma
propagation in micrometric flexible capillaries and plasma splitting in branching
geometries is presented and second, the first feasibility and tolerance studies dealing
with NTP delivery in colon and lung of anesthetized mice was performed. These are
the preliminary successful experiments dealing with NTP endoscopic applications.
The impact of plasma propagation on the surrounding tissues before reaching the
tumor environment has not yet been characterized, and will be one of the key issues
to implement a beneficial plasma endoscopic treatment. Plasma selectivity on different
tumor cells would be of great importance to address this problem.
Acknowledgements The authors are grateful to the Region Centre (APR Plasmed) and ANR
2010 BLAN 093003 PAMPA financial supports. MV is supported by Germitec, VS by Conseil
Gnral 45, DR by CNRS and Rgion Centre.
References
30.1 Introduction
One of the most exciting medical discoveries of the 1980s in the study of human
physiology was the realization that nitric oxide (NO) is a short-lived, endogenously
produced molecule that acts as a signaling molecule in the body. NO is a gas with one
unpaired electron and thus is a free radical (NO) that reacts with many biological
molecules. The discovery of signal transmission by a NO gas, produced by one cell,
which penetrates membranes and regulates the function of other cells was awarded by
Noble Prize in Physiology and Medicine for 1998 and was considered as an entirely
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 393
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_30, Springer Science+Business Media B.V. 2012
394 V.N. Vasilets and A.B. Shekhter
new principle for signaling in the human organism. NO has important roles in the
function of many tissues and organs, from the cardiovascular system to the brain [1].
Physiology and biochemistry of NO have been discussed in more than 40,000 papers
since 1990. Wound healing processes are known to involve a sharp increase of nitric
oxide generation in wound tissue. This increase is caused by the activation of constitu-
tive isoforms of NO-synthase (eNOS and nNOS) and by a markedly enhanced synthe-
sis of inducible NOS (iNOS) [2]. These observations suggest a possible therapeutic
use of various NO donors for the acceleration of the wound healing. Our previous
studies [3] indicated that gaseous NO flow produced by air-plasma generator Plason
acts beneficially on the wound healing. To understand the mechanism of this effect the
NO levels in the tissues of rats were studied using exogenous NO spin traps, Fe2+
complexes with diethyldithiocarbamate (DETC)1 or N-methyl-D-glucamine dithio-
carbamate (MGD). NO binding with these traps results in the formation of paramag-
netic mononitrosyl iron complexes, MNICDETC or MNICMGD, which are soluble
in lipophilic or hydrophilic media, respectively [4]. As a result of these experiments
the sharp increase of MNICDETC and nitrosylhemeiron complexes in wound
tissue as a response to successive short-lasting exposures to gaseous NO flow. It was
shown that endogenous NO is responsible for the formation of these complexes, and
that this NO is synthesized by NOS enzymes. The EPR experiments gave direct proof
that gaseous NO treatment increased the stationary level of endogenous NO molecules
in the wound tissues. We propose that this beneficial effect could be caused by the
mechanism involving peroxynitrite as an intermediate. Small doses of peroxynitrite
may actually have protective action in cell cultures and tissues. These results were
attributed to a mobilization of various antioxidant defenses by the presence of per-
oxynitrite [5]. This mobilization could diminish the amount of superoxide anions in
wound tissue, thereby protecting endogenous NO molecules against superoxide attack
By implication, more endogenous NO molecules become available as signalling mol-
ecules to regulate the metabolic processes in wound tissue. Thus, the therapeutic effect
of the exogenous NO treatment is attributed to enhanced NO bioavailability.
Today NO is known also as a universal anti-microbial factor. The sterilization effect
of NO is proved for more than 45 bacteria including gram-negative, gram-positive,
spore-forming rods, alpha-hemolytic anaerobe, facultative anaerobe, cocci arranged in
clusters, anti-biotic-resistant bacteria as well as biofilms (S aureus) and other infections
[611]. Nitric oxide is also a very effective agent against fungi [1113] and parasites
(Leshmaniasis) [1416]. One of the main benefit of NO bio-decontamination is that no
identification of infecting microbe is necessary since NO treats all pathogens. It is
important to mention also that the topical antibacterial market for broad spectrum
agents that effectively kill drug resistant bacteria is >$1.4B only in US.
Fig. 30.1 Multi-purpose medical plasma chemical source Plason. (a) configuration in hot
mode applications (b) configuration in cold mode applications
Fig. 30.2 Manipulators for destruction (1), therapy mode (generation of room temperature
NO-containing gas flow) (2), coagulation, wound sterilization (3)
Fig. 30.3 Experimental concentrations of NO and NO2 depending on the distance from manipulator
1 (therapeutic concentrations 300500 ppm)
Table 30.1 Results of stimulation for neutral and radical particles in DC arc
discharge in air at the temperature 3,000 K
Species N2 O2 H2O Ar H
mol% 6.7E + 01 9.1E + 00 1.5E-01 8.9E-01 1.8E + 00
Species N O OH NO H2
mol% 1.8E 02 1.5E + 01 1.3E + 00 4.9E + 00 1.8E 02
the two-step cooling system, gas channels of which are created in a maze scheme to
force-cool the jet by the liquid circulating from the cooling system. This construc-
tion allows for obtaining NO-containing gas flow with sufficiently low temperature,
and optimal concentration of nitric oxide molecules, which makes it possible to
apply this manipulator for treatment of external body surfaces by using the cooling
hose of 150 mm length (temperature of NO at the exit ~36C). Of course, NO con-
tent in the gas flow depends on the distance from the exit channel (Fig. 30.3).
Additionally, for laparoscopic operation, a special manipulator of 350 mm length
and 10 mm diameter is utilized (Fig. 30.2, manipulator 2).
The possible operating regimes of the apparatus are defined by the characteristics
of the gas flow exiting from the manipulator, main parameters of which are its tem-
perature and the nitrogen oxide content. The typical concentration of NO used for
wound healing and sterilization varies in the range 300500 ppm (see Fig. 30.3).
The device Plason generating active species in air plasma was approved for
medical purposes by Russian Ministry of Health in 2000 and has been used for
medical applications in Russia more than 10 years for the treatment of various dis-
eases associated with inflammatory and destructive processes, regeneration distur-
bances and vascular dysfunctions. In 2009 Plason was also certified in Czech
Republic as medical device Class IIb (EC Certificate No 09 0637 QS/NB) and suc-
cessfully applied by the company ONKOCET in Slovakia [19].
398 V.N. Vasilets and A.B. Shekhter
Since the discovery of antibiotics there has never been a time when there has been a
greater need for a new antimicrobial platform. The main reasons of this emergency
situation are:
the emergence of multi-drug resistant bacteria
the continued inappropriate use of antibiotics in humans and in animals
newer antibiotics are scarce and are often far more expensive and more toxic than
predecessors
there may soon be diseases once easy to cure that become untreatable
the announcement that payors will no longer cover hospital acquired infections
Bacterial Skin and Soft Tissue Infections (SSTI) are also a rapidly growing prob-
lem, with ever-increasing morbidity, due to the growing incidence of multi-drug
resistant bacteria. Over 22 million SSTIs were reported in 2009. In the US, there
were 15.6 million ambulatory visits and over 6.5 million non-ambulatory visits for
SSTIs, an increase of more than 100% from 2001 [11].
Nitric oxide novel approach uses a variation of the bodys normal defense against
pathogens, which delivers fast action and universal spectrum coverage, at a dose
that is lethal to all bacteria and fungus but is fully tolerated by human cells. NO is
used by the body to kill bacterial, viral, fungal and parasitic infections. The GSH
pool protects the bacteria from the attack by NO and H2O2. NO and H2O2 are two
endogenous mechanisms of pathogen control. (NO being released by macrophages,
GSH = Reduced Glutathione). In each case, the defense mechanism is based on
physiological NO levels. Our approach is to locally overwhelm the infection with
supra-physiological levels of NO which has a very short lifetime (69 s). Human
cells since they secrete NO to kill naturally have a much higher tolerance for NO
and are either minimally impacted or stimulated.
In vitro investigation of the NO bio-decontamination effect on Escherichia coli,
Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, gram-negative
Pseudomonas aeruginosa, gram-positive methicillin-sensitive Staphylococcus aureus,
Clostridium difficile and Candida albicans, which are typically associated with many
hospital infections, showed that treatment by NO with the concentrations 200400 ppm
practically inactivate them all [611]. NO could be used also to kill antibiotic-resistant
(methicillin-resistant Staphylococcus aureus, MRSA), fungal (Tinea Pedis,
Coniothyrium minitans) [79] and parasitic infections (Leishmaniasis) [11, 1416].
In vivo investigation of NO anti-bacterial properties was done in animal experi-
ments using Plason plasma source. Two sets of experiments were carried out with
90 male rats. In the first set of experiments conditioned aseptic skin wounds (300 mm2)
were inflicted. A rings were inserted in the wound edges and covered with cello-
phane to prevent drying. After 2nd, 4th, 6th and 8th days wound surface and edges in
first experimental group (25 rats) were treated with NO-containing (300 ppm) flow
from Plason for 30 s (distance 20 cm, T = 40C). In the control (25 rats) the wounds
were exposed only to the warm air at the same temperature. In the second set of
30 Nitric Oxide Plasma Sources for Bio-Decontamination and Plasma Therapy 399
30 s; control) 3
500 ppm 300 ppm
0
0 3rd 7th 14th
experiments (40 rats), wounds were infected with Staphylococcus aureus culture
(109 bacteria per sample) for simulation of infected suppurative wound. The experi-
ment and the control were exactly the same as in the first set. But 20 rats were treated
with NO concentration 300 ppm and the other 20 rats were exposed by 500 ppm NO
concentration. On days 3rd, 7th, 14th and 21st the bacterial content in the wounds
was measured using traditional colony forming unit (CFU) method.
For the model of conditioned aseptic full-thickness plane wounds (Fig. 30.4.),
nitric oxide treatment (4 sessions, 30 s, 300 ppm) reduced bacterial level in the
experimental group up to 7% CFU/cm2 (more than ten times) in comparison with
the control group where the reduction was limited to 43%. The anti-bacterial effect
of NO-containing plasma gas was much more pronounced for the model of infected
(Staphylococcus aureus), full-thickness plane wounds (Fig. 30.5). The reduction of
CFU/cm2 value more than four orders of magnitude was observed for 300 ppm NO
concentration and about five orders of magnitude sterilization effect was obtained
for 500 ppm NO treatment procedure on the 21st day. For comparison the reduction
of bacteria load for the control group was about two orders of magnitude less than
that for 500 ppm NO-treatment (Fig. 30.5).
400 V.N. Vasilets and A.B. Shekhter
Concluding this part of the paper one can say that nitric oxide treatment has the
profile to become a first-line treatment for all skin and soft tissue infections, whether
caused by bacteria, fungus, or parasites.
30.5 Conclusions
could be enhanced by the synergy effects of NO/H2O2 and NO/O2 are known in the
regulation of cell apoptosis, antimicrobial macrophage defense processes and treat-
ment of primary pulmonary hypertension [2328].
Therefore, plasma NO-therapy is a promising technique for treatment and pre-
vention of inflammatory, ulcerative, necrotic and sclerotic processes as well as
vascular dysfunctions in the human body.
References
1. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology and
pharmacology. Pharmacol Rev 43:109142
2. Schaffer MR, Tantry U, Barbul A (1997) Nitric oxide metabolism in wounds. J Surg Res
71:2531
3. Shekhter AB, Kabisov RK, Pekshev AV, Kozlov NP, Perov IL (1998) Experimental clinical
substantiation of plasma dynamic therapy of wounds with nitric oxide. Bull Exp Biol Med
(Rus) 126:210215
4. Serezhenkov VA, Vanin AF, Shekhter AB, Rudenko TG, Gavrilchak AV, Pekshev AV (2001)
Dynamics of the levels of endogenous and exogenous nitric oxide in wound and organ tissues
of rats: EPR assays. In: NO-therapy: theoretical aspects and clinical experience of exogenous
nitric oxide application in medicine (Moscow): 2735
5. Laude K, Thuillez C, Richard V (2002) Peroxynitrite triggers a delayed resistance of coronary
endothelial cells against ischemiareperfusion injury. Am J Physiol 283:H1418H1423
6. Kerr AR, Wei XQ, Andrew PW, Mitchell TJ (2004) Nitric oxide exerts distinct effects in local
and systemic infections with Streptococcus pneumoniae. Microb Pathog 36:303310
7. Morita H, Yoshikawa H, Suzuki T, Hisamatsu S, Kato Y, Sakata R, Nagata Y, Yoshimura T
(2004) Anti-microbial action against verotoxigenic Escherichia coli O157: H7 of nitric oxide
derived from sodium nitrite. Biosci Biotechnol Biochem 68:10271034
8. Satriano J (2004) Arginine pathways and the inflammatory response: interregulation of nitric
oxide and polyamines: review article. Amino Acids 26:321329
9. Fox S, Wilkinson TS, Wheatley PS, Xiao B, Morris RE, Sutherland A, Simpson AJ, Barlow
PG, Butler AR, Megson IL, Rossi AG (2010) NO-loaded Zn2+-exchanged zeolite materials: a
potential bifunctional anti-bacterial strategy. Acta Biomater 6:15151521
402 V.N. Vasilets and A.B. Shekhter
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 403
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_31, Springer Science+Business Media B.V. 2012
404 P. Luke et al.
31.1 Introduction
Li et al. [15, 16] observed the mechanical rupture of intracellular gaseous vacuoles
of cyanobacteria cells exposed to underwater streamer discharge, which was caused
presumably by the physical effects of shock waves induced by the discharge.
Underwater shock waves are also used in extracorporeal shockwave lithotripsy
(ESWL) to non-invasively disintegrate kidney stones and gallstones. In fact, ESWL
is one of the most successful applications of shock waves in medicine. Since the
mid-1980s, lithotripsy has been in clinical use worldwide for treating kidney stones
and continues to be the favored method for uncomplicated, upper urinary tract cal-
culi, even with the advent of percutaneous surgical methods. The treatment involves
focusing shock waves generated by the ESWL device (lithotripter) outside of the
patients body to disintegrate the stone at a depth in tissue. The targeted stone is
located at the shock wave focus, and hundreds to thousands of shock wave pulses
are delivered to comminute the stone. Acoustic coupling with the patient is achieved
through water baths or liquid-filled pillows. After the urinary stone treatment, the
stone debris passes through the urinary tract. In the case of gallstones, the stones are
chemically dissolved. A typical pressure waveform at the lithotripter focus in water
consists of a leading shock wave front (compressive wave) with a peak positive
pressure in the range of 30150 MPa and a phase duration of 0.53 ms, followed by
a tensile wave with a peak negative pressure down to 20 MPa and a duration of
220 ms [17].
Over 40 models of clinical lithotripters are commercially available worldwide
(electrohydraulic, electromagnetic and piezoceramic). In electrohydraulic litho-
tripters, an underwater high-current spark discharge between a pair of electrodes is
generated at the focus of the ellipsoidal reflector, and the emerging spherical shock
wave produced by the plasma at the spark gap is concentrated on the kidney stone
located at the second focus of the ellipsoid [18]. At the end of the 1980s, one of
modified versions of the electrohydraulic type of lithotripter was developed at the
Institute of Plasma Physics AS CR [1921]. These generators are used in the litho-
tripters Medilit (produced by the company MEDIPO, Brno, Czech Republic) at
approximately 20 hospitals in the Czech and Slovak Republics [22]. So far, more
than 120,000 patients have been successfully treated with these devices.
The success of the ESWL stimulated research on the applications of focused
shock waves (FSW) in other branches of medicine. Shock waves are used in orthopedy
for the fracture healing, the treatment of pseudarthrosis, tendinopathy and other
orthopedic diseases [23]. Great attention has been on to the role of cavitation in
ESWL-induced biological effects on soft tissues and the possible treatment of some
types of cancers. It was initially studied to identify possible side effects of the litho-
tripsy therapy, such as haematuria, renal colic, perirenal and intrarenal hematomas,
pancreatitis and arrhythmias [17]. The evidence suggests that cavitation is an impor-
tant factor in lithotripsy. Stones initially break by spall, and then erosion grinds the
fragments into a size suitable for the patient to pass [18]. Micro-inhomogeneities or
microbubbles in the fluid (e.g., water or urine) that are located in the vicinity of the
focal zone may produce cavitations, which may interact with the stone but also with
the cells/tissue. The threshold for cavitation in water is approximately 0.5 MPa; how-
ever, this depends on the duration of the tensile pulse [17].
406 P. Luke et al.
Figure 31.1 shows the scheme for a generator of focused tandem shock waves in
water. The generator is divided by an acoustically transparent membrane (Mylar
foil, thickness 100 mm) into two parts. The first part, which is filled with a highly
conductive saline solution (on the order of tens of mS/cm), consists of two compos-
ite metallic cylindrical high-voltage electrodes of different diameter d and length h
(A1: d = 90 mm, h = 17 mm; A2: d = 60 mm, h = 55 mm) placed along the axis of the
outer metallic parabolic reflector. The composite anode consists of a metallic cylin-
drical electrode covered with a thin porous ceramic layer deposited by thermal
plasma spraying. The role of the ceramic layer is to redistribute the electric field on
the electrode during the pre-discharge phase. Due to the differences in conductivity
and permittivity between the water and the ceramic layer, the electric field strength
on the surface of the composite electrode is many times enhanced in comparison
with a metallic electrode of the same dimensions. This setup allows for the simulta-
neous generation of a large number of filamentary discharge channels distributed
almost homogenously around the entire surface of the composite electrode at a
moderate applied voltage [36].
A pulsed high voltage of positive polarity was applied to the composite elec-
trodes using a pulse power supply that consists of two capacitors (C1, C2)
charged up to 30 kV and two triggered spark gaps (SG). The spark gap switches
were triggered either at the same time (switch S was closed) or with a variable
delay (switch S was open), allowing us to produce either a single shock wave or
two successive shock waves focused to a common focal point (i.e., focused tandem
408 P. Luke et al.
Fig. 31.1 The scheme of the generator of focused tandem shock waves in water
shock waves) with an adjustable delay between the waves. The spark gaps were
triggered by 12 kV/ms pulses produced by discharging the capacitors through the
thyratrons. The thyratrons were triggered by a dual-channel pulse generator with
a variable time delay (100 ns step) between the channels. The focal point of the
parabolic reflector is situated in the second part of the generator, which is filled
with tap water and is located 5 cm from the Mylar membrane. Each discharge
channel formed at the composite electrode creates a semi- spherical pres-
sure wave in the saline solution. By superposition of the waves, a cylindrical
pressure wave propagating from the anode is formed. The primary cylin-
drical pressure wave is focused by a reflector. Near the focus, this wave is
transformed into a strong shock wave. The amplitude and, to some extent, the
waveforms of the shock waves, at the focus can be controlled by the anode
surface areas, the discharge circuit parameters and the solution conductivity,
which play a decisive role in the characteristics of underwater plasmas. Higher
31 Generation of Focused Shock Waves in Water for Biomedical Applications 409
conductivity leads to higher power density in the discharge channel, and this
results in an increase in the plasma electron density, a higher plasma temperature
and more intensive pressure wave evolution from the discharge [32]. In this work,
saline solution conductivity of 18 mS/cm was used for the generation of the dis-
charge at the composite electrode, which was based on results from previous
work that demonstrated that the given experimental setup generates the highest
amplitude of the pressure wave at this conductivity [33].
Schlieren photography was used to acquire time-resolved images of the pressure
field induced by the shock wave(s) in the focal region. The Nd:YAG pulsed laser beam
[wavelength = 532 nm, pulse width = 10 ns full width at half maximum (FWHM)] was
expanded to a diameter of 60 mm and applied through a focal region as an illumina-
tion light source in a perpendicular direction to the shockwave propagation. A camera
was set up to acquire the Schlieren photographs of the focal region at various time
delays after the discharge ignition by the triggered spark gap switch SG1 and synchro-
nized with the firing of the laser beam via a delay generator. Consequently, the pres-
sure field and the pressure waveforms of the shock waves produced at the focus were
measured by polyvinylidene fluoride (PVDF) shock gauge sensors (Model S25_04,
Piezotech SA, France). The pressure field at the focal region was mapped point-by-
point, and the focal volume with the dimensions 2.5 mm 32 mm long (FWHM)
was determined [32, 33]. For observation of the dynamics of the cavitation at the focal
region, a microscope and a PCO 12-bit charge-coupled device (CCD) Sensicam cam-
era were used. Microscope magnifications of 10 to 25 were used. The focal area
was illuminated by a xenon lamp, which was fed by a high-voltage capacitor switched
by a thyratron. The ignition of the lamp was synchronized with the triggered spark gap
switch SG1 at various time delays. The exposure time of the PCO camera was 400 ns.
This exposure time was sufficient to obtain sharp and bright photos.
B16 melanoma syngeneic cells were grown in a tissue culture medium (6 106
cells). The viability of the B16 melanoma cells after exposure to FTSW was exam-
ined using the trypan blue staining method and by optical imaging microscopy.
Tumor growth delay experiments were performed with Lewis strain rats inoculated
subcutaneously with R5-28 malignant cells (2.5 105 cells/rat) [37]. The time of the
tumor growth latency and the volume of the growing sarcoma in the Lewis rats were
followed throughout the experiment and compared with the control (not exposed)
tumors (four animals in each group). The tumor size was measured with Vernier cali-
pers. The tumor volumes V at particular time intervals were calculated by the formula
V = a b2 p/6, where a and b are the tumor length and tumor width, respectively.
FTSW-induced lesions in rabbits thigh muscles were examined by magnetic reso-
nance imaging (MRI). Prior to exposure, the rabbits were anesthetized and depilated
on their abdomen. The scanning procedure was carried out on an MR tomograph
Siemens Magnetom Trio 3T at the Institute of Clinical and Experimental Medicine,
Prague, Czech Republic. The echo (TE) and repetition (TR) times were set as fol-
lows: TE = 20 ms and TR = 642 ms. The slice thickness of the MRI scans was set to
2 mm. The imaging procedure was carried out on the first, second and seventh day
after the exposure to shock waves. The rabbit was under anesthesia during imaging.
410 P. Luke et al.
The pressure field at the focal region formed by the focused tandem shock waves
was visualized by Schlieren photography. Figure 31.2 shows the pressure field for
three different situations. Figure 31.2a, b show the pressure field when only the first
or the second waves were generated, respectively. The waves propagate from right
to left in these pictures. The white cross-like spot in the picture specifies the region
of the highest pressure of the focused shock wave with a sharp front of the shock
waves. The convergence angle corresponds to the reflector geometry and the posi-
tion of the composite electrode in the reflector. The circle in the picture indicates a
secondary short-wavelength spherical shock wave created by the collapsing cavita-
tion bubbles. Figure 31.2a, b show that both shock waves, if produced separately (or
with a long time delay between them), generated only a small number of cavitations
at the focus. Figure 31.2c shows the situation when two successive waves interacted
(time delay of 15 ms). The second wave is heavily damped in the focal region, and
this created a very complex pressure field with a large number of secondary short-
wavelength shock waves from the collapsing cavitation. A shock wave duration of
approximately 70 ns and a corresponding wavelength on the order of 0.1 mm were
estimated, which are capable of interacting with cell-scale structures.
Figure 31.2 demonstrates that the degree of interaction of the second shock wave
with the first one depended on the time delay between the waves. Figure 31.3 shows
the pressure wave-forms of two interacting waves arriving at the focus with differ-
ent time delays td. These measurements indicate that there are three different phases
in the interaction. The first and second phases are demonstrated in Fig. 31.3a.
A relatively short delay between the shocks (time interval of 815 ms) resulted in the
second pressure wave creating a large amplitude rarefraction wave at the focus
instead of an originally positive pressure wave. With increasing time intervals
between the waves, the amplitude of the second shock wave rapidly decreased. For
the interval 50 ms < td < 500 ms, the second shock wave was totally damped. The
second shock started to propagate as a pressure wave for td > 600 ms, and its
propagation was fully re-established only at time delays above 1 ms (Fig. 31.3b).
Fig. 31.2 Schlieren photographs of the focal region: (a) first shock wave only, (b) second shock
wave only, and (c) two interacting shock waves. The time interval between the waves was 15 ms.
The direction of the shock wave (SW) propagation was from the right to the left (as depicted by
the arrow)
31 Generation of Focused Shock Waves in Water for Biomedical Applications 411
Fig. 31.3 The pressure waveforms of the two interacting shock waves measured at the focus for
different time delays between the first and the second shock waves
Fig. 31.4 The dynamics of cavitations at the focal region after the interaction of the focused tan-
dem shock waves for a time delay of 10 ms between the waves
This scenario can be explained by assuming that the cavitation bubbles formed by
the first shock wave served as cavitation nuclei for the second shock wave.
Because the cavitation nuclei dissipate over time, the interval between the shock
waves affected the total number of cavitations and the amplitude of the negative
pressure. Figure 31.3a shows that the largest interaction between the first and second
wave was obtained for a time delay of 815 ms, resulting in the maximum negative
phase of the pressure wave of second shock wave and a large number of cavitation.
With increasing time delay between the waves, the second wave propagated through
the perturbed me-dium, and its energy was absorbed by the collapsing cavitations.
Therefore, the influen-ce of the first shock wave on the second wave was insignifi-
cant at longer time delays (td > 600 ms) when most of the cavitations produced by
the first wave were quenched.
An example of the evolution of the cavitation bubbles formed in water at the focus
after the interaction of the focused tandem shock waves for a time delay of 10 ms is
shown in Fig. 31.4. The growth of the bubbles lasted for about 200250 ms (reaching
a maximum bubble radius ~1.3 mm), and then the bubbles began to collapse.
412 P. Luke et al.
The presented FTSW generator was used to investigate the biological effects of
focused tandem shock waves. A time delay between the waves of 10 ms was used
based on previous experiments, which showed that the largest interaction of the
waves occurred at this condition (i.e., td = 815 ms). The amplitude of the pressure
(positive) wave was 80 MPa with a duration of 0.7 ms. The amplitude of the rarefac-
tion (negative) wave was 80 MPa with a duration of 2 ms. These waves produced a
large number of cavitations at the focus (see Figs. 31.2, 31.3, and 31.4). The dimen-
sion of focal volume was 2.5 mm 32 mm long (FWHM) [32, 33]. The applied
voltage was 30 kV. The charging capacity was 0.8 mF. The pulse repetition fre-
quency of the applied tandem shock waves was 0.7 Hz.
Figure 31.5 shows optical micrographs of melanoma B16 cells exposed in vitro
to a different number of tandem shocks. Polypropylene Eppendorf vials with a sus-
pension of melanoma cells (1.5 ml) were placed at the focus of the generator and
exposed to the shock waves. Figure 31.5 demonstrates that exposure of carcinomas
cells to the tandem shocks resulted in cell membrane perforation and total damage
(fragmentation) of the cells at a higher dose. The subsequent analysis of the cell
viability of the B16 melanoma cells confirmed greater damage to the cells with an
increasing number of applied tandem shock waves (data not shown).
Consequently, effects of focused tandem shocks on soft tissue were examined
in vivo. Figure 31.6 shows MRI images of a targeted lesion in a rabbits thigh mus-
cle caused by applying 900 tandem shocks and its progress for 7 days after FTSW
exposure. The left side of each image is a control showing the non-exposed muscle.
The lesion in the tissue is clearly visible as a dark spot of hematoma surrounded by
a bright area of accompanying edema (marked by the white arrow). The differences
in the shape and size of the hematoma and edema over time can be seen. After
1 week, the hematoma disappeared, and the edema area was reduced, indicating
recovery of the injured tissue in the shock wave impact area. The larger size of the
hematoma (~ 5 mm) compared to the focal area of the FTSW (~ 2.5 mm) can be
explained by the changes in the acoustical properties of the treated tissue with an
increasing number of applied shocks, which leads to enhanced damage of the tissue
at the targeted area by the shock waves. Additional MRI analysis revealed that the
extent of the damage in the direction of the shock propagation is about 15 mm. The
subsequent histological examination of the injured femoral muscle showed an
extensive focus of granulation tissue in the field of subacute dystrophic changes in
the muscle fibers with a very sharp transition between the damaged and non-exposed
tissue (not shown). These results showed that tandem shocks are capable of causing
localized lesions at a predictable location deep within soft tissue at the focus without
damaging tissue located in front of the focal point. This finding is important in terms
of the potential biomedical applications of tandem shock waves, such as in treat-
ment of tumors that are inoperable or very difficult to be treated (e.g., many liver
metastases and pancreatic tumors).
31 Generation of Focused Shock Waves in Water for Biomedical Applications 413
Fig. 31.5 Optical micrographs of the melanoma B16 cells exposed in vitro to different numbers
of focused tandem shock waves: (a) control, (b) 200 shocks and (c) 400 shocks
Fig. 31.6 MRI images of targeted lesions in a rabbits thigh muscle (marked by the white arrow)
over time induced in vivo by applying 900 focused tandem shock waves. The left side in each
image is a control showing the non-exposed muscle. (a) 1st day, (b) 2nd day, and (c) 7th day after
FTSW exposure
Figure 31.7 shows the in vivo effect of FTSW on the growth of sarcoma tumors
in rats of the Lewis strain. R5-28 malignant cells of the syngeneic sarcoma were
subcutaneously applied caudally on the right and left sides of Lewis rats and
tumors were allowed to grow for 35 days, after which the tumors were treated by
FTSW. The tumors on the left sides of the rats were exposed to 240 shocks by
placing the anesthetized animals in the water bath of the FTSW generator. The
contralateral tumor served as a control. Shock waves were applied to the tumors
from two different angles with 120 shock waves at each angle. Figure 31.7 shows
that, in comparison with the intact controls, the growth of the exposed tumors was
significantly delayed.
Considering the tumor size and much smaller size of the shock wave focus area
(~ order of magnitude difference), the scanning of the tumor by shock waves can be
414 P. Luke et al.
used to increase the efficiency and to enlarge the localized effect of shock wave
exposure. Varying the number of applied shocks can also be used. In our case, a
higher dose (~400 shocks) resulted in mechanical damage to the tumor tissue. Thus,
repeated treatment of the tumor by a smaller number of shocks over several days
might be considered. Apart from mechanical effects, the shock waves may also
contribute to tumor treatment due to cavitation-induced sonochemical effects by the
intracellular formation of radicals. Combining the effect of shock waves with
anticancer drugs may also be possible (e.g., to enhance the chemotherapy efficiency
by increasing the permeability of tumor cells membranes to cytostatic drugs by the
shockwaves or through the sonodynamic activation of cytotoxic drugs by cavitation
induced by FTSW). Research on the possible synergetic effects treatment with
shock waves in conjunction with other therapeutic methods used in cancer treatment
is in progress.
31.5 Conclusions
Acknowledgements This work was supported by the Czech Science Foundation (project No.
202/09/1151) and the Ministry of Education, Youth and Sports of the Czech Republic (MSM
0021620808). The authors would like to thank Dr. V. Horak from the Institute of Animal
Physiology and Genetics AS CR for providing R5-28 malignant cells for the experiments with the
Lewis rats, Dr. V. Herynek and Dr. M. Dezortova from the Institute for Clinical and Experimental
Medicine, Prague, Czech Republic for the MRI analysis of the lesions in the rabbits thighs and
Dr. J. Kralova from the Institute of Molecular Genetics AS CR for optical micrographs of the
melanoma B16 cells.
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Chapter 32
DBD Plasma Assisted Silver Functionalization
of Surgical Meshes
32.1 Introduction
Surgical mesh can be seen as a reinforcement or patch, which is inserted to the weak
spot or defect in the patient tissue. The wound is healed after the surgery in a way
where the mesh acts as an external support through which the new tissue is grown,
J. Rhe (*)
Faculty of Science, Masaryk University, Kotlsk 2, 61137 Brno, Czech Republic
Department of Experimental Physics, Comenius University, Mlynsk dolina F2,
Bratislava, Slovakia
e-mail: rahel@mail.muni.cz
H. Polkov E. Jonov
Faculty of Science, Masaryk University,Kotlsk 2, 61137 Brno, Czech Republic
M. Hudcov P. Nasadil
Textile Testing Institute, Vclavsk 6, Brno, Czech Republic
M. Zahoran
Department of Experimental Physics, Comenius University, Mlynsk dolina F2,
Bratislava, Slovakia
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 417
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_32, Springer Science+Business Media B.V. 2012
418 J. Rhe et al.
providing a tension-free repair [1]. The most common types of surgical meshes are
hernia mesh, stress urinary incontinence slings and mesh for treating prolapse.
Hernias are very common medical condition which affects during the life time
510% of the population. The use of polymeric surgical meshes for hernia repair
was introduced by Francis Usher, who in 1958 began promoting the use of polyeth-
ylene and polypropylene meshes (commercial name Marlex) [1, 2]. Nowadays
lightweight knitted meshes of pore size larger than 1 mm are used preferably in
order to improve the biocompatibility by reduced amount of foreign tissue [3].
Polypropylene (PP) is the most widely used polymer for the manufacture of sur-
gical meshes. It is a cheap material offering a proper tension strength and high level
of chemical inertness, which is responsible for its extreme resistance to biological
degradation and high level of biocompatibility. Polyester (PES) meshes are less
frequent than polypropylene. An example is a multifilament Mersilene (Ethicon) or
CHS 100 (VUP). PES is a flexible, well adaptive mesh, but it has a higher risk of
infection and cause a greater degree of inflammatory response.
Infection is among one of the most serious complications associated with the inser-
tion of implant into the body [4]. According to Engelsman et al. [5] surgical mesh
implant is prone to infiltration by bacteria even 39 months after insertion into the
body. Gram-positive S. aureus, S. epidermidis and gram-negative E. coli are the most
common bacteria infecting surgical meshes [4]. Experiments showed that the multi-
filament implants are due to greater surface more susceptible to bacterial colonization
than monofilament [4, 6].
The risk of bacterial infection can be substantially reduced by an appropriate mesh
surface modification [5, 6]. Successful results on antimicrobial surface finishes of
meshes were achieved using various chemical compounds such as silver, gold, tita-
nium, chlorhexidine, triclosan, chitosan [4, 79]. Scheindbach et al. [10] studied influ-
ence of titanium on the surface of hard polypropylene mesh (titanium-coated Atrium).
Positive effect on the biocompatibility and the reduction of bacterial adhesion was
demonstrated. Saygun et al. [11] studied the effect of gold and gold with palladium
coatings. A significant reduction in bacterial colonization was shown, in particular
when combining gold and palladium.
Silver and its various forms (silver oxide, silver sulfadiazine, silver carbonate,
silver nitrate, silver iodide, etc.) is probably the most widely used antimicrobial
metal agent. Silver can be readily combined with other substances, which may
increase and prolong the desired antibacterial effect. Fox et al. [12] lists in his patent
various combinations of antimicrobial agents, focused primarily on the synergistic
effect of silver salts and chlorhexidine or chlorhexidine salts.
Table 32.1 summarizes the composition and functional finishing for some of the
most common commercially available PP and PES meshes. Surprisingly neither of
them employs silver as an antimicrobial agent. To our knowledge the only commer-
cially available surgical mesh employing silver is DUALMESH PLUS (WL Gore
and Associates) which is made from rather expensive expanded polytetrafluoroeth-
ylene (ePTFE). Its antimicrobial action is facilitated by the combination of silver
carbonate and chlorhexidine [4, 13]. The mesh is claimed to be resistant to bacteria
for 10 days after its implantation.
32 DBD Plasma Assisted Silver Functionalization of Surgical Meshes 419
reducing agents may be provided in the form of organic radicals created in the
suitably chosen solution by ionizing radiation, which is already proved method in
making metal nanocolloids [18]. A favorable energy balance then drives reduced
zero valent atoms to mutually dimerize or associate with the excess of ions which
eventually leads to the progressive coalescence into silver clusters Agmz+ with
nuclearity m and number of associated ions z.
In the following text we will present the results of our work on preparing silver
particles functionalized surgical meshes made of PP a PES and the evaluation of
their performance. To reduce the final cost of process being developed, surface
activation involving DBD at atmospheric pressure was adopted. Our study is focused
primarily on PP meshes functionalization, which is not only commercially more
important product, but due to its well-known chemical inertness it represents a
particular challenge for chemical surface modification.
32.2 Experiment
Volume dielectric barrier discharge (DBD) transparent electrode system was assem-
bled from two water filled transparent polycarbonate blocks, separated by two
dielectric barriers plates made of 1.1 (0.05) mm thick borosilicate glass D263 T
(er = 6.7, suppl. by Praezisions Glas and Optik, Germany). The particular combina-
tion of low surface roughness and high permittivity value of D263 T was found to
be beneficial to stabilizing DBD in its diffuse mode of operation.
The inter-electrode gap was adjusted by 3 screws located underneath of the bottom
electrode to 0.7 mm. Electrode system was housed in a transparent gas-tight reactor, to
allow gas containment control. The reactor was operated at atmospheric pressure in
flowing gas at q = 4 dm3/min. N2 of technical purity and compressed air were used. The
reactor was purged with the working gas for 2 min prior any treatment run.
The active dimensions of the electrodes were 130 128 mm. Regular tap water
(specific conductivity 100 mS/cm) was used simultaneously both as an electric con-
ductor and as a cooling medium. The whole experimental set-up for plasma treat-
ment is shown in Fig. 32.1.
The electrodes were energized by a sinusoidal voltage from a VF 1500 ozone
generator power supply, made by Lifetech (Czech Rep.). The maximum operating
power was 300 W, operating frequency was 203 kHz. Two TEKTRONIX P6015A
(1,000) high-voltage probes were used to control applied voltage to electrodes.
Pearson current monitor, model 4100 (1:1, 35 MHz, 10 ns usable rise time), was
used for the electrical current measurements. Visual appearance of the plasma was
recorded using digital camera CASIO EX-F1 (6.0 Mpx). Signals from the HV
probes and current monitor were recorded using a LeCroy WaveRunner 6100A
oscilloscope (2 GHz Bandwidth, 10 GS/s max sample rate).
Fig. 32.1 Experimental setup: MOD T2 water filled electrodes, HV high voltage probe, CM cur-
rent monitor, Ck variable capacitor, OSC oscilloscope, PC computer, GVF power supply, CIRC
water circulation system, CCD digital camera
was used to prepare primary silver deposit. Water solutions of NaOH, Na2CO3 and
NaCl with concentrations of 1 or 0.5 M were used to transform primary silver
deposit into silver salts of Ag2O, Ag2CO3 and AgCl respectively. Concentrated
HNO3 was employed for preparing the titration extract from AgNO3, Ag2O and
Ag2CO3. Titration extract from AgCl was prepared by 5% sodium thiosulfate. All
chemical used were of p.a. purity.
Qualitative testing of the antibacterial effect was done according to the ISO
20645 standard [26]. The test is based on the assessment of bacterial growth around
and under the textile specimen that was laid into the particular bacterial strain
inoculated agar. The test will result in three possible outcomes for antimicrobial
activity insufficient effect (bacterial growth in agar under the specimen), limit of
efficacy (growth almost totally suppressed, restricted to small colonies); good effect
(no bacterial growth underneath the specimen, presence of inhibition zone around
the specimen). The purpose of test is to identify the minimum concentration of
antibacterial agent in order to achieve final assessment of good effect.
Gram-positive S. aureus ATCC 6538 and gram-negative E. coli ATCC 11229
were evaluated. Cultivation took place on plate count agar (PCA) and nutrient agar
(NA). Ten milliliters of pure, sterile agar was poured into plastic Petri dishes and let
to solidify. Afterwards 5 ml of 45C bacteria inoculated agar was added (1 5.108 CFU/
ml). This temperature was found to be sufficient for keeping agar in a flowing state
without killing bacteria. Mesh specimen was laid into the inoculated agar. After incu-
bating samples for 24 h at 37C the evaluation was carried out by the assessment of
bacterial growth under the tested specimen and by the size of inhibition zone around
the specimen. Each cultivation test was made for two separate samples.
Quantitative analysis of amount of immobilized silver was done by the potentio-
metric titration. Orion 4-Star Benchtop pH/ISE meter (Thermo Fisher Scientific)
equipped with the PerfectIONTM combination Ag/S electrode (Mettler-Toledo) was
used. Silver coated polymer meshes were immersed for 1 h into concentrated HNO3
(65%) at 60C to dissolve immobilized silver. Consequently the solute was diluted
32 DBD Plasma Assisted Silver Functionalization of Surgical Meshes 423
to approx 1:10 by deionized water and titrated with the water solution of NaCl in the
absence on light. To verify the obtained data, the concentration of silver in solute
was determined also by atomic absorption spectrometry using UNICAM AAS 939
spectrometer. Flame atomizer or graphite tube electrothermal (ETA-AAS) atomizers
was used for high and low concentration of Ag respectively. The difference between
both methods was found be less than 20%, which is within the stated accuracy of
used AAS spectrometer.
Morphology of silver particles immobilized of meshes was studied by SEM
using VEGA II SBH (fy TESCAN, Brno, Czech Rep.) offering the resolution of
2 nm. Prior the SEM analysis all samples were coated by gold using magnetron-
sputtering device.
The use of transparent DBD reactor revealed immediately poor contact of generated
plasma with individual mesh fibers when treating in air gas (Fig. 32.2a). DBD
microfilaments were formed preferentially in relatively large inter-fibrous spaces.
To address this issue plasma treatment using N2 diffuse DBD was adopted.
Figure 32.2b illustrates the visual uniformity of N2 diffuse DBD in contact with PES
fiber. The improved treatment efficiency was confirmed by shortening of time nec-
essary for complete water wettability of polymer mesh to approx. half of the time
needed in air. In the following text all plasma activation were made in N2.
Immediately after plasma treatment samples of PES and PP meshes were
immersed into light-tight PP Erlenmeyer flask filled with AgNO3 water solution of
various concentration. The solution with samples was magnetically stirred at
250 rpm for 24 h at room temperature. After removing from the solution samples,
the excess of water in inter-fibrous space was shaked off and samples were let to dry
at the room temperature.
Table 32.2 summarizes the results of bacterial efficacy for various initial concen-
tration of AgNO3 solution for 2 min lasting plasma treatment. The formation of the
inhibition zone around the Ag functionalized PES sample is apparent from Fig. 32.3.
Initial AgNO3 concentration of 0.05 M was found to be sufficient to provide
desired antibacterial effect for both PES and PP meshes. The actual relative mass of
immobilized silver from this solution was measured to be (6.0 0.1) mg/g for PES
mesh and (3.8 0.1) mg/g for PP mesh. Smaller amount of Ag immobilized on PP
can be attributed to its higher chemical inertness and smaller fiber surface area (see
Fig. 32.4). It is interesting to note, that it was possible to approximately double
these values when the immersion time in AgNO3 solution is cut from 24 to 1 h. The
results obtained then were (14 3) mg/g for PES and (5.0 0.4) mg/g for PP mesh.
The exact nature of competing process responsible for the removal of immobilized
silver back to the solution has not been identified yet.
424 J. Rhe et al.
Fig. 32.2 Plane view on plasma treated PES samples of 4 4 cm: (a) DBD in air; (b) Diffuse DBD
in N2. exposure time 1 s for both pictures
The positive effect of plasma treatment was obvious for both PES and PP samples.
PES sample immersed to AgNO3 without prior plasma treatment resulted in the
silver relative mass of (3.3 0.6) mg/g. This is only 55% of Ag mass immobilized
on plasma-activated samples. The mass of 3.3 mg/g corresponds well with the mass
of Ag present in fluid adhering to the mesh immediately after its removal from the
deposition flask. Our conclusion is that solid state Ag compound on untreated PES
is formed by precipitation during the course of water evaporation (drying).
Plasma activation of chemically more inert PP samples resulted in similar rela-
tive enhancement although smaller in absolute numbers. We were able to increase
the absolute amount of Ag by extending the exposure time in N2 DBD plasma. The
results obtained were: (1.8 0.1) mg/g for untreated PP mesh; (3.8 0.1) mg/g for
2 min treatment; (5.8 0.2) mg/g for 4 min treatment and (7.7 0.3) mg/g for 10 min
lasting plasma treatment.
32 DBD Plasma Assisted Silver Functionalization of Surgical Meshes 425
Fig. 32.3 The result of ISO 20645 cultivation test for E. coli inoculated agar: (a) Heavy growth
under the untreated PES mesh insufficient effect; (b) No growth under the PES 0.01 M mesh and
the growth inhibition zone of 1 mm around the mesh good effect
Fig. 32.4 Morphology of Ag2O coating on investigated polymer meshes. View field of 100 mm for
both photographs
where R* denotes an active site on the polymer surface. We assume that this
process is occurring in two steps. In the first step is the Ag+ reduction to
zero-valence state on the polymer surface radical. In the second step excess of
NO3 present in the solution contributes to its conversion to solid precipitate of
AgNO3. Nevertheless more detailed experiments on the process kinetics still
need to be done.
Table 32.3 Bacterial efficacy against E.coli and S. aureus of various Ag salts on PP mesh
E. coli S. aureus
(Ag)/(PP) Inhibition Inhibition
Sample [mg/g] zone [mm] Assessment zone [mm] Assessment
PP untreated 0 0 Insufficient 0 Insufficient
PP + AgNO3 3.8 0.1 1 Good 2 Good
PP + Ag2O 5.3 0.7 1 Good 1 Good
PP + Ag2CO3 1.5 0.5 1.5 Good 2 Good
PP + AgCl 2.0 0.5 1 Good 1 Good
SEM magnified view of Ag2O deposited on PP and PES meshes are shown in
Fig. 32.4. The same qualitative pictures were obtained also for the rest of investi-
gated functionalizations. In the case of PES submicron crystals of Ag salts are
deposited preferentially in areas between adjacent filaments of yarn. This prefer-
ence may turn be of practical importance, as bacteria in our cultivation experiments
are colonizing preferentially exactly the same locations. For PP silver salts are
deposited directly on the monofilament surface with relatively large degree of het-
erogeneity. We can observe spots completely covered with grown microcrystal, as
well as areas sparsely covered with submicron monocrystals. Further optimization
of deposition process could certainly address the observed heterogeneity; neverthe-
less as was shown in Table 32.3 even such heterogeneous surface is able to provide
sufficient antibacterial action.
428 J. Rhe et al.
32.4 Conclusion
References
1. Pandit AS, Henry JA (2004) Design of surgical meshes an engineering perspective. Technol
Health Care 12:5165
2. Read RC (1999) Francis C. Usher, herniologist of the twentieth century. Hernia 3:167171
3. Hollinsky C, Sandberg S et al (2008) Biomechanical properties of lightweight versus heavy-
weight meshes for laparoscopic inguinal hernia repair and their impact on recurrence rates.
Surg Endosc 22:26792685
4. Engelsman AF, van der Mei HC et al (2007) The phenomenon of infection with abdominal
wall reconstruction. Biomaterials 28:23142327
5. Engelsman AF, van der Mei HC et al (2008) Morphological aspects of surgical meshes as a
risk factor for bacterial colonisation. Br J Surg 95:10511059
6. Engelsman AF, van Dam GM et al (2010) In vivo evaluation of bacterial infection involving
morphologically different surgical meshes. Ann Surg 251:133137
7. Carbonell AM, Matthews BD et al (2005) The susceptibility of prosthetic biomaterials to
infection. Surg Endosc 19:430435
32 DBD Plasma Assisted Silver Functionalization of Surgical Meshes 429
Abstract In this review the potential applications of cold atmospheric gas plasmas
are presented with particular reference to the problems of contamination of foods
by biological agents. In addition to the accidental contamination of food, the very
real threat arising from the deliberate contamination of the human food chain is
also considered. The evidence that has been gained for the efficacy of cold plasmas
in inactivating a wide range of biological agents is briefly surveyed. This is fol-
lowed by an examination of previous work in which various types of foodstuffs
have been successfully treated using cold gas plasmas. The need to demonstrate
that the quality attributes of treated foods is not adversely affected is stressed.
Finally, the role which gas plasmas may have in decontaminating food processing
equipment is considered.
33.1 Introduction
The World Health Organisation (WHO) claims that 1.5 million children under five
years of age die each year due to diarrhoea primarily as a result of contaminated
drinking water and food. WHO also estimates that globally there are about two
billion cases of diarrhoeal disease annually [50]. This is not a phenomenon con-
fined to the developing world; Mead et al. [24] stated that in the United States
there were 76 million incidences of foodborne disease resulting in 323,000 hospi-
talizations and 5,200 deaths annually. Moreover, this pattern is repeated in all
G. Shama (*)
Department of Chemical Engineering, Loughborough University,
Ashby Road, LE11 3TU Loughborough, Leics, UK
e-mail: g.shama@Lboro.ac.uk
M.G. Kong
Department of Electronic and Electrical Engineering, Loughborough University,
Ashby Road, LE11 3TU Loughborough, Leics, UK
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 433
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_33, Springer Science+Business Media B.V. 2012
434 G. Shama and M.G. Kong
industrialized countries. In England and Wales in the year 2000 for example, there
were 1,338,772 cases of foodborne infections resulting in 20,759 hospitalizations
and 480 deaths [2].
Increasingly more of the food we consume is subject to processing of one sort or
another. Some form of processing must inevitably take place immediately following
harvest or slaughter, but in industrialized countries food has become subject to ever
more processing to meet the changing demands of consumers for convenience
foods. There is mounting evidence to show that so-called ready to eat (RTE) foods
such as fresh cut salads for example are increasingly becoming associated with
incidences of foodborne disease [38]. This trend towards increased consumption of
convenience foods is inevitably influencing the way in which the food industry pro-
duces such products. Production is increasingly becoming centralised into fewer,
but larger, processing facilities. This in itself increases the impacts which isolated
incidences of contamination of food products may have on the population as a
whole. A telling example of this is an incident associated with the US dairy indus-
try; a Salmonella outbreak in a single product liquid ice cream resulted in some
224,000 people [41] being affected. A more recent example concerns the Kellogg
company. Salmonella-infected peanut butter obtained from one of the companys
suppliers became incorporated into some of its snack products. In the ensuing epi-
demic of foodborne disease that followed 714 persons became infected in 43 states
and there was evidence to suggest that infection from these food products may have
been a contributory factor in 6 deaths. Moreover, the incident led to the recall of
seven million cases of Kelloggs products [7].
Whilst there have been improvements in the microbiological safety of foods,
few, if any foodborne pathogens have been contained over a sustained period of
time. Bovine spongiform encephalitis (BSE) is still being detected in cattle, and
the possibility cannot be entirely ruled out that BSE might have infected sheep fed
the same contaminated feed supplements that caused the epidemic in cows [15].
Even apparent successes against particular agents hold little long term comfort; an
increase in salmonellosis took place throughout the 1980s that was largely due to
Salmonella Enteritidis Phage Type 4 which was found predominantly in poultry
and eggs. Concerted action was taken in a number of countries which involved the
wholesale slaughter of infected flocks coupled with extensive programmes of
immunisations, and this proved successful as the incidences of this disease showed
a significant decline during the 1990s. However, there are indications that since the
year 2000 incidences are once more on the increase. The culprits are now different
phage types of S. Enteritidis, and it appears that as one pathogen is effectively
removed from a particular niche others evolve to take its place [27].
Another recent concern regarding food is the potential target it offers to terrorist
organisations. Although Manning et al. [23] cite a source claiming that since 1912
there have only been 12 documented cases of deliberate attempts to introduce patho-
genic agents into foods or livestock, the current perceived threat level remains high.
A further consequence of the centralisation of food processing is that it serves to
increase the impact which terrorist attacks directed towards the food supply chain
33 Prospects for Treating Foods with Cold Atmospheric Gas Plasmas 435
might have. Although there have been no concerted recent attempts made against
national food supplies, examples exist that serve to expose both the vulnerability of
the food supply chain and provide an indication of what the consequences of the
deliberate contamination of elements of the supply chain could be. This is illus-
trated by an incident of contaminated Worcester sauce that occurred in the UK in
2006. One of the ingredients that was added to a batch of the sauce was chilli pow-
der to which had been added a dye, Sudan Red I. The dye is not approved for food
use and is in fact a Category 3 carcinogen. The sauce subsequently became widely
distributed in a very large number of prepared foods and when the (deliberate) con-
tamination of the chilli powder was discovered the UK Food Standards Agency
ordered the withdrawal of over 600 different products from retail outlets [16]. The
motives in this case were financial rather than political or ideological but the exten-
sive permeation of a single ingredient throughout a nations food chain should serve
as a salutary warning.
Another area of increasing concern in the food industry is the removal of traces
of allergens from food processing facilities and equipment. If this could be achieved
it would allow different products to be processed using the same equipment thus
affording processors both versatility in the way they process foods and also cost
savings. According to a review by Kagan [19] between 1% and 2% of adults and
between 5% and 7.5% of children in the United States are subject to food allergies
of one sort or another. The food industry as a whole expends a great deal of effort in
attempting to remove allergens from the surfaces of food processing equipment and
has also to label its products using a rather convoluted system based on terms such
as may contain, free from, suitable for which are apt to confuse consumers and
may ultimately lead a proportion of them to take risks [48]. In essence this attests to
the difficulty of assuring total removal of allergens.
In what follows below the emphasis will be on the potential that atmospheric,
non-thermal gas plasmas have for the treatment of food in order to ensure its micro-
bial safety. Reference to the challenges that have to be faced for this to be achieved
will also be made as required. In addition to foods themselves, it is appropriate to
consider the environments and the equipment used during the processing of food.
The emphasis here will be on the opportunities for the application of plasma tech-
nology rather than on the technology itself. For the latter the excellent recent review
by Ehlbeck et al. [13] is strongly recommended.
There are about 200 known agents that are capable of causing disease when ingested
[1]. These include chemical, physical and microbial agents. The latter comprise bac-
teria, fungi, parasites, viruses and prions and allergens. To date all of the biological
agents that have been tested have proved themselves susceptible to the effects of
436 G. Shama and M.G. Kong
plasma treatment. Naturally with reference to foods the aim must be to maximise the
damage inflicted on the disease-causing agents whilst minimising any deleterious
impacts on the food itself. The latter are quite complex assessments to make, but include
damage to key nutrients, or changes in e.g. the texture or appearance of the food.
An understanding of the mechanisms by which gas plasmas bring about death or
lethal injury to pathogenic agents could potentially lead to the enhancement of
future treatment strategies through the manipulation of plasma conditions to maxi-
mise inactivation of pathogens. Perhaps the most-studied organisms to date have
been the bacteria. Both Gram positive and Gram negative bacteria, biofilm-formers
and bacterial spores have all been shown to be inactivated by gas plasmas [11, 29,
31, 44]. It is also amongst this group of organisms that the greatest body of knowl-
edge is being accumulated regarding mechanisms of inactivation. Moisan et al. [25]
attempted to infer information about possible mechanisms of inactivation of Bacillus
subtilis spores by examining the form of survival curves and SEMs of spores post
treatment. These workers concluded that UV photons played a significant role in the
inactivation of the spores and were aided in this by oxygen atoms. Later, Perni et al.
[30] used E. coli mutants to show that the inactivation of these bacteria was primar-
ily due to oxygen atoms and that the contributions of UV photons and OH radicals
were relatively minor. Although these accounts might appear contradictory, the
experimental data upon which they are based was conducted with different organ-
isms, using different plasma systems and under different conditions. It will evi-
dently be some time before the mechanism of inactivation of living cells can be
unified. However, studies such as those cited above will hopefully continue to pro-
vide useful information and allow deeper insights into the principal mechanisms.
Moving away from bacteria, gas plasmas have also been used to inactivate fungal
spores [45] and more recently, Avramidis et al. [3] were able to show that plasmas
caused deformation and disruption to the hyphae of growing fungi belonging to the
genera Ascochyta and Fusarium. Previously Perni et al. [31] had demonstrated the
plasma-induced inactivation of yeast of the species Saccharomyces cerevisiae.
Parasites have received rather less attention from the gas plasma community.
Two important pathogens are Giardia lamblia and Cryptosporidium parvum [40].
These organisms form oocysts frequently simply referred to as cysts that are
somewhat akin to bacterial and fungal spores in being able to withstand environ-
mental hardships. Both are common waterborne organisms that resist chlorination
the currently employed methods for disinfecting water.
The number of foodborne incidents due to viruses is less well-documented owing
to the difficulties in detecting viruses in foods. Notwithstanding, Roberts and
Antonoplos [35] were able to show that a hydrogen peroxide gas plasma steriliza-
tion unit was effective against a range of viruses including HIV type 1, hepatitis A
virus, respiratory syncytial virus, vaccinia virus, herpes simplex virus type 1, and
poliovirus type 2. Using a similar system, Vickery et al. [43] obtained total inactiva-
tion of duck hepatitis virus as a model for human hepatitis B virus. Of the viruses
selected in these studies, only hepatitis A is a foodborne virus, but the broad range
of viral types inactivated by gas plasma suggests that other foodborne viruses such
as Norovirus would also be susceptible to plasma treatment.
33 Prospects for Treating Foods with Cold Atmospheric Gas Plasmas 437
The term biological agent not only includes living organisms but also their
components and their products. One important category in this regard is the various
types of proteins produced by living organisms. Deng et al. [12] demonstrated the
ability of gas plasmas to both destroy and physically remove a protein, bovine serum
albumin (BSA), from the surface of stainless steel. This suggests a potential role for
gas plasmas in inactivating prions which are the proteins responsible for bovine
spongiform encephalopathy (BSE) and variant Creutzfeldt Jacob disease, (vCJB).
More recently, Bayliss et al. [5] used a pulsed radio frequency plasma jet to bring
about the destruction and removal of amyloid fibrils from mica sheets. The former
are viewed as a realistic non-infectious prion model.
The results obtained using these model proteins also suggest that it may be pos-
sible to remove food allergens from the surfaces of food processing plant. The food
processing industry has been obliged to take seriously the possibility of inducing
allergenic reactions in the population at large and has adopted an approach based
around that of Good Manufacturing Practices (GMP) that aims to assure segregation
of allergenic ingredients and declaration of the possible existence of allergens as was
mentioned above. Current decontamination practices centre on conventional clean-
ing techniques using detergents and sanitizers coupled with in some cases swab-
based assays based on the detection of adenosine triphosphate (ATP) which can be
used as a proxy indicator of the presence of protein on surfaces [47].
The most obvious application of gas plasmas in the food industry would be to
employ them directly to decontaminate foods. Evidence has steadily been accumu-
lating to show that gas plasmas can be used to effect reductions in microbial counts
for a wide range of different foods covering virtually all food types including meat,
dairy products and plant foods such as fruits, salad crops and nuts. A compilation of
such results for a range of foods is shown in Table 33.1.
Whilst the number of foods that are being subjected to plasma treatment contin-
ues to increase and in many cases results in acceptable reductions in bacterial counts,
a significant proportion of the studies listed in the table did not take into consider-
ation whether treatment resulted in any deleterious effects in the food. This would
include changes that might affect the quality, or the nutritional value, or even the
appearance of the foodstuff itself. The exceptions to this statement are the investiga-
tions of Vleugels et al. [44] with bell peppers, Basaran et al. [4] on various types of
nut, Niemira and Sites [28] on apples, Ragni et al. [34] on shell eggs, and Kim et al.
[20] for bacon. It cannot be stressed how important such considerations are as the
chemical species produced in the plasmas possess the potential of bleaching the
natural colour of foods and/or damaging key nutrients such as vitamins. Treatment
could conceivably result in the peroxidation of lipids in fatty foods. Kim et al. [20]
attempted to conduct assays to establish whether plasma-treated bacon showed evi-
dence of peroxidation but their results were inconclusive. In addition to what might
438 G. Shama and M.G. Kong
be termed chemical effects, the gas flows used in the plasmas may result in
moisture losses from the food undergoing treatment. These effects can all be quanti-
fied, but the ultimate test is consumer acceptance which needs to be assessed using
specially trained panels.
Perhaps the next most important application of plasmas would be in the treatment
of food processing equipment. Micro-organisms that attach to the surfaces of such
equipment have been shown to cause the cross contamination of foods that subse-
quently come into direct contact with the contaminated surface [9]. Arguably the
most notorious case being that of the epidemic of typhoid caused in Aberdeen,
Scotland in the summer of 1964. A catering-sized can of corned beef from
S. America contaminated with Salmonella was sliced using a food slicing machine
in a grocers premises in Aberdeen. The contaminated blade resulted in the cross
contamination of other food products subsequently sliced using the machine and
this resulted in an epidemic in which 500 people were hospitalised and a virtual
national state of panic [39]. This mechanism of microbial transfer has been investi-
gated in studies conducted by Sheen and Hwang [37] for ham slicing and by Perni
et al. [32] for fresh fruit cutting. Leipold et al. [22] proposed an innovative solution
to this problem. They designed a dielectric barrier discharge (DBD) plasma system
in which the circular cutting blade of a food slicing machine constituted one of the
electrodes. With this they were able to demonstrate the efficient inactivation of
Listeria innocua sprayed onto the blade. This represents a most interesting approach
to the concept of decontaminating food processing machinery and points the way to
extending the concept to other operations such as conveying etc.
An unconventional approach to food preservation was taken by Fernandez-
Gutierrez et al. [14] who attempted to coat the surface of apples with vanillin using
a plasma-based thin film deposition technique to protect the food against fungal
spoilage. The conditions chosen by these researchers did not result in a continuous
coating but rather in what they referred to as nodules. The concept is however an
interesting one and could be potentially applicable to other foods.
Another potential area where plasmas might make an impact is in food packag-
ing. Although relatively little work appears to have been conducted using atmo-
spheric gas plasmas, Heise et al. [17] demonstrated the efficacy of DBD against
bacterial and fungal spores deposited onto polymer foils.
The physical environment in which food processing takes place can occasion-
ally become colonised by micro-organisms that appear to survive cycles of
decontamination treatments and which are referred to as resident organisms [33].
This may occur because some organisms are only exposed to sublethal concentra-
tions of chemical disinfectants and thus are able to develop resistance against them
[46]. Although as stated above, the precise mode of action of gas plasmas has yet to
be fully elucidated, it seems likely that more than one plasma species will be
involved. This would lessen the possibility of organisms developing resistance.
The threats to the safety of foods resulting from potential terrorist action were
briefly mentioned above. Certain sectors and elements of the food industry have been
identified as being particularly vulnerable. Wein and Liu [49] for example constructed
a mathematical model to predict the consequences of the deliberate release of
440 G. Shama and M.G. Kong
botulinum toxin into the milk supply chain in California, USA. The worst possible
case envisaged the poisoning of up to some 5 105 people but this required a substan-
tial quantity of toxin (of the order of 100 g). Nevertheless, the implications are unset-
tling. A possibly more readily available toxin is ricin. Ricin is a potent toxin that is
found in the castor oil plant. Its threat lies in part from the fact that the plant grows
widely throughout the world and that it is relatively easy to extract from the castor
bean mash that remains after pressing for oil. Jackson et al. [18] studied the thermal
inactivation of ricin in reconstituted infant formula. They found that the some of the
thermal treatment regimes currently in place were insufficient to inactivate the toxin.
Existing measures to safeguard foods against microbial contamination may therefore
not be sufficient to render harmless such toxins. Could plasmas be used to provide
protection against such toxins? The literature would appear to indicate that the use of
gas plasmas in this context has not been investigated, and therefore this warrants some
attention by researchers in the field. A possible indication that they could is provided
by the work of Birmingham and Hammerstrom [6] who showed that plasmas possess
the ability to destroy a mycotoxin in aerosolised form. However, considerably more
work would be needed to extend their findings to the cases mentioned above.
The range of foods subject to plasma treatment will doubtless continue to grow.
If gas plasmas are ever to escape the confines of the laboratory and assume accep-
tance in the food industry a number of factors aside from their proven ability to
inactivate pathogens, will need to be taken into account. This would include exhaustive
testing to ensure that foods that have been treated with plasmas do not undergo any
undesirable changes that would render them harmful, unpalatable or otherwise
unsaleable. Absolute proof that no harmful products have been produced is actually
very difficult, if not impossible, to demonstrate as the list of potential toxic by-products
is a very long one. Ensuring the quality of treated foods is a rather more straightfor-
ward undertaking. This would include tests for key nutrients such as vitamins, tex-
tural studies, colour etc. The issue of public perception of foods treated with gas
plasmas is also one that needs addressing. Ill-informed publicity could impact
negatively on the chances of this technology being adopted by the food industry.
Mention was made above of the increasing popularity of RTE foods and the
problems associated with minimally processed foods such as salads etc. One assess-
ment of the current state of affairs is that the available technology for disinfection
has not kept pace with the changing eating habits of consumers and that an effective
and adaptable new decontamination technology is sorely needed. Adaptability will
be essential, as not only must the technology prove itself efficacious against current
pathogens, but it will need to provide assurance that it can operate equally effec-
tively against emerging pathogens. Climate change will constitute one driver of
change. That it will influence global food production can surely not be in doubt
[36], more uncertain however will be its impact on food pathogens. Increases in
33 Prospects for Treating Foods with Cold Atmospheric Gas Plasmas 441
temperature will have some direct effects including increasing the growth rate of
most of the common foodborne pathogens. But it will also influence eating behav-
iour. Warmer temperature are associated with increased consumption of salads and
barbecued foods both of which have been heavily implicated in incidences of
foodborne disease [21].
One assessment of the true scale of the problem faced by any decontamination
technology may be gained from a telling statement from the work of Newell et al.
[27]; over millennia all food-borne pathogens have developed efficient and effec-
tive strategies, which exploit, wholly or in part, food as a vehicle to transfer from
one human gut host to another, or from an animal to a human. The mechanisms
involved are complex and varied but all are able to survive intervening periods in the
environment, and then avoid the human innate gut defences to colonize and multi-
ply rapidly before enabling effective dispersal, frequently through fluid faeces, back
into the environment to progress again through each cycle.
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26. Moon SY, Kim DB, Gweon B, Choe W, Song HP, Jo C (2009) Feasibility study of the steriliza-
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Chapter 34
Decontamination of Bacillus subtilis Spores
in a Sealed Package Using a Non-thermal
Plasma System
Abstract The safety of packaged food and medical devices is a major concern to
consumers and government officials. Recent inventions (PK-1 and PK-2) based on
the principles of non-thermal, atmospheric plasma has shown significant reduction
in bacterial contamination inside a sealed package.
The objective of this study was to evaluate the PK-1 and PK-2 systems in the
reduction of Bacillus subtilis spores using packages containing air or modified
atmosphere (MA) gas (65% O2/30% CO2/5% N2). The experimental design con-
sisted of the following parameters: (1) two voltage conditions: 13.5 kV with 1.0 cm
electrode gap (PK-1) and 80 kV with 4.5 cm electrode gap (PK-2), (2) two treatment
conditions: inside and outside the field of ionization, (3) PK-1 and PK-2 optimized
treatment times: 300 and 120 s, respectively, and (4) two package gas types: air and
modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). Measurements included:
(1) bacterial reductions of Bacillus subtilis var. niger (B. atrophaeus), (2) ozone,
nitrous oxides (NOx), and carbon monoxide concentrations, and (3) relative humidity.
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 445
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_34, Springer Science+Business Media B.V. 2012
446 K.M. Keener et al.
Bacillus subtilis (1.7 106/strip) were loaded into sterile uncovered petri dishes and
treated with ionization generated in packages using air or MA gas blend. Samples
were treated for 300 s (PK-1) or 120 s (PK-2) and stored at room temperature for
24 h. Results documented relative humidity (RH) ranged from 20% to 30%. After
300 s of PK-1 treatment (13.5 kV/44 W/1.0 cm gap), ozone concentrations were
6,000 ppm (air) and 7,500 ppm (MA). After 120 s of PK-2 treatment
(80 kV/150 W/4.5 cm), ozone concentrations were 7,500 ppm (air) and 12,000 ppm
(MA). Ozone and NOx concentrations were non-detect (ND) after 24 h. PK-1 car-
bon monoxide levels were <20 ppm (air) and <100 ppm (MA) after 24 h. The PK-2
carbon monoxide levels were <20 ppm (air) and <400 ppm (MA) after 24 h.
Treatments showed reductions in spores of greater than 6 log10 after 24 h. Reductions
were maintained without additional re-growth at 72 h. These results indicate that the
PK-1 and PK-2 systems have the capacity to reduce Bacillus subtilis spores in an
in-package ionization process.
34.1 Introduction
Air (78% N2, 22% O2) and modified atmosphere (MA) gas (65% O2, 30% CO2, 5% N2)
were purchased from a local gas supplier at specified concentrations with a certificate
of analysis. These gas composition(s) were then metered into sealed package at a rate
of 2.1 L/min. using a flow meter (Model 2260, Gilmont Instruments, Inc., Barrington,
IL, USA) yielding final fill volume of 1.5 L with average fill time of 45 s.
Clear, 3.78 L Ziploc (SC Johnson and Son, Inc., Racine, WI, USA) heavy duty
freezer bags were obtained from a local grocery store. Bags consisted of low-density
polyethylene (LDPE) and were 1.6 mm thick. All treatments were double bagged
for storage time of 24 h at room temperature.
Bacillus subtilis var. niger (B. atrophaeus) spore strips (NAMSA, Northwood, OH,
USA) with size of 3.2 cm 0.6 cm, each containing Bacillus populations of 1.7 106/
strip or 6.23 log10 were loaded into open sterile petri dish inside treatment package
448 K.M. Keener et al.
and then used in ionization treatments. For infield ionization with PK-1 system, one
end of each spore strip was secured with transparent tape to the inside of the storage
bag within electrode gap space prior to treatment.
Treatments were carried out utilizing either the PK-1 system developed in the Food
Technology Development Laboratory at Purdue University or the PK-2 system
(Phenix Technologies, Accident, MD). The PK-1 system is capable of generating
ozone inside a sealed package at various geometries and is a patent-pending tech-
nology [8]. The PK-1 system was operated at 44 W and 60 Hz generating 13.5 kV
of potential between the electrodes (1.0 cm gap). Electrodes consisted of coils of
wire wound around a dielectric base with a treatment area of 92.4 cm2
(7.7 cm 12 cm). The PK-2 system was operated at 150 W and 60 Hz generating
80 kV of potential between the high voltage circular stainless electrodes (15 cm
diam., 4.5 cm gap) with polypropylene (pp) insulation layers: top (11.1 mm) and
bottom (3.2 mm) between electrodes. Treatment of all samples occurred at room
temperature. The storage bags containing spore samples were filled with the work-
ing gas (air or MA) and purged three times to ensure purity of the gas in the bag.
Electrodes were placed above and below the bag, oriented directly above each other
to allow for maximum ionization of plasma field. Electrodes rested on top of each
other with the bag in between having an approximate gap distance of 1.0 cm (PK-1)
and 4.5 cm (PK-2). Hollow plastic spacers were inserted inside the bag to maintain
proper gap (PK-1).
Temperature of the electrodes and treated storage bags was measured prior to and
immediately after treatment using an infrared thermometer (Omega Engineering,
Inc., Stamford, CT, USA). Temperatures of treated samples registered at room tem-
perature for both ionization systems immediately after treatment. The electrodes of
the PK-1 system were allowed to cool (2530C) for 300 s between treatments for
uniform treatment temperature conditions.
Ozone and nitric oxide concentrations were measured immediately following the
300 or 120 s treatments as well as after 24 h storage. This was completed by using
34 Decontamination of Bacillus subtilis Spores in a Sealed Package 449
Spore recoveries and aseptic methods were followed per manufacturer (NAMSA,
Northwood, OH, USA) for population verification of Bacillus subtilis spore strips.
After ionization treatment and 24 h storage, each strip was aseptically removed
from bag and transferred into sterile 20 150 mm test tube containing 10 mL of
0.1% sterile peptone. Ten sterile 6 mm glass beads were then added to each test
tube. Each test tube was vortexed (model vortexer 59, Denville Scientific, Inc.,
Metuchen, NJ, USA) on high speed for 120 s or until the spore strip was fully mac-
erated into loose fibers. Test tubes were then heat shocked in order to fully germi-
nate spore population by placing into a 500 mL beaker with 300 mL of water heated
to 90C and maintained at 8085C for 10 min. Test tubes were transferred to a cold
tap water bath momentarily (2 min), and then to ice water bath to rapidly cool test
tubes to 04C. Test tubes were then removed from ice bath and further serial dilu-
tions were performed including 102, 103, 104, and/or 105 based on treatments or
recoveries of positive (+) controls (Bacillus populations of 1.5 106/strip, 6.18
log10). The required aliquot volumes from corresponding serial dilutions were then
plated into respective petri dishes (100 15 mm) containing sterile Tryptic Soy Agar
(TSA) prepared per Difco Manual specifications for spore colony enumeration [11].
TSA plates were incubated at 3031C and colony growth and recoveries were
monitored at 24, 48, and 72 h.
450 K.M. Keener et al.
Optical emission spectral analysis was performed using the PK-1 System
(12.0 kV). The plasma emission was captured via a 0.22 NA optical fiber and
analyzed using a low-resolution UV-VIS spectrometer operated in the wavelength
range 2001,000 nm. The optical fibre was aligned at the centre of the plasma at
a distance of 6 cm.
Relative humidity and temperatures inside the storage bags were measured using a
Springfield Precise Temp relative humidity sensor (Taylor Precision Products,
Oak Brook, IL, USA) recorded at 0 and 24 h storage.
Bacillus subtilis populations and gas concentrations were analyzed using t-test
comparisons (P < 0.05) and standard deviations in Microsoft Excel (2010 Version).
Figure 34.1 shows the optical emission spectra for an air plasma obtained with the
PK-1 system operating at 12 kV. The spectra clearly shows emission data at wave-
lengths previously characterized for air plasma species as noted by other researchers
[4]. The species include N2, N2+, O2+, and O (see table of assignments for the main
peaks as an insert in Fig. 34.1). The spectra for MA gas was similar (data not shown)
with the same major peaks. Interestingly, there were no further differences noted;
although reactive oxygen species generation rates were significantly different
between air and MA gas.
Figures 34.2 and 34.3 document the reactive oxygen species generation during
in-package ionization at the specified times for both 13.5 and 80 kV. It can be seen
from these data that high levels of reactive oxygen species can be generated for both
air and MA gas. However, reactive oxygen species generation rates were signifi-
cantly different between air and MA gas. At 13.5 kV an ozone generation rate of
1,200 and 1,500 ppm per minute were observed for air and MA gas, respectively.
At 80 kV an ozone generation rate of 3,750 and 6,250 ppm per minute were observed
for air and MA gas, respectively. These results suggest that ionization voltage
increases reactive oxygen species generation rate, even at constant voltage gradient.
In air, the nitrous gas concentrations did not significantly change with ionization
34 Decontamination of Bacillus subtilis Spores in a Sealed Package 451
336 nm N2
357 nm N2
380 nm N2
390 nm N2+
405 nm N2+
426 nm N2+
673 nm O2+
686 nm O2+
776 nm O
Fig. 34.1 Optical emission spectroscopy of air plasma generated using PK-1 ionization system
(12.0 kV)
Fig. 34.2 Gas concentrations generated using PK-1 ionization system (13.5 kV)
voltage. Both voltages (13.5 and 80 kV) achieved maximum nitrous gas concentration
of approximately 1,000 ppm. Conversely, the MA gas nitrous gas concentrations
reached a significantly higher level with increased ionization voltage. Nitrous gas
concentrations at 80 kV reached over 4,000 ppm at 120 s treatment.
452 K.M. Keener et al.
Fig. 34.3 Gas concentrations generated using PK-2 ionization system (80 kV)
Both ozone and nitrous oxides levels decayed to zero within 24 h of treatment.
However, there was a measureable carbon monoxide concentration in MA gas at
24 h post-treatment with levels 200 and 400 ppm for the 13.5 and 80 kV at treatment
times of 300 and 120 s, respectively. As stated in Material and Methods, the current
carbon monoxide measurement technique does not allow measurement in presence
of ozone (i.e., time zero).
Figures 34.4 and 34.5 illustrate the spore reductions with ionization treatment.
It is observed that in-package ionization both inside and outside of the ionization
field at 13.5 and 80 kV can eliminate Bacillus subtilis spores. At 13.5 kV, treatment
times for MA gas spore elimination were 180 and 300 s for outside and inside field,
respectively. At 13.5 kV, treatment times for air spore elimination were 300 s out-
side of the ionization field with insignificant spore reductions (<1.2 log) inside the
ionization field. Similar results were reported by researchers utilizing a sealed enve-
lope containing Bacillus cereus spores and air in a DBD system operating at 30 kV
at 1.3 kHz [12]. At 80 kV, complete elimination of spores was shown in 15 s or less
with no measureable difference in spore reduction rates between air and MA gas.
It was observed that the inside the field, high voltage treatment times did show
slightly increased spore populations recoveries at 48 h compared to 24 h; however,
no additional organisms were recovered at 72 h. These results demonstrate that
using an 80 kV in-package ionization process, air or MA gas can provide complete
34 Decontamination of Bacillus subtilis Spores in a Sealed Package 453
Fig. 34.4 Spore reductions generated using PK-1 ionization system (13.5 kV)
Fig. 34.5 Spore reductions generated using PK-2 ionization system (80 kV)
454 K.M. Keener et al.
elimination of Bacillus subtilis spores in less than 15 s or less. For these studies, dry
air was used; and all samples were maintained at between 20% and 30% relative
humidity at room temperature. It is expected that elevated humidity may provide an
even greater spore reduction rate.
34.4 Conclusions
References
35.1 Introduction
Traditional methods for surface decontamination of fruits are based on the use of
chemical biocides such as chlorine and washing procedures [1]. In order to reduce
the use of the chemical products, the water, the chemical residuals in the environment,
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 457
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_35, Springer Science+Business Media B.V. 2012
458 A. Berardinelli et al.
and to improve the treatment efficacy and postharvest quality, new physical methods
are currently being researched. The application of UV radiations [2] and gaseous
ozone [3] are undoubtedly the most studied technologies for fresh fruit and vegetables.
Non-thermal gas plasma could also represent an alternative to the conventional
methods used for fruit surface decontamination. Non-thermal or cold plasma, char-
acterized by an electronic temperature much higher than the macroscopic one, can
be produced at atmospheric pressure and by using air as working gas [4]. These
conditions make the technique suitable for food decontamination when the quality
of the products must be preserved.
Non-thermal atmospheric plasma can be generated by means of AC or DC power
supply, by microwaves or by RF sources and the choice of the frequency exciting
mode influences the plasma characteristics (electrons and heavy species behaviour)
[5]. The dielectric barrier discharge (DBD), the resistive barrier discharge (RBD) and
the atmospheric pressure plasma jet (APPJ) are the most researched methods [6].
During the last years several researches were conducted on the use of cold plasma
for the decontamination of food products. The DBD method was successfully used
on shell eggs deliberated inoculated with Salmonella Enteritidis and S. Typhimurium;
negative effects of the treatment were not observed both on the eggshell and in the
internal components [7]. The lethal efficacy of the cold atmospheric plasma was
also investigated on surface cut sections of different types of fruit and vegetables
previously inoculated with food-borne pathogens (Escherichia coli, Salmonella and
Listeria monocytogenes) and spoilage organisms (S. cerevisiae, P. agglomerans and
G. liquefaciens) [8, 9]. Experiences on the entire vegetable surface were conducted
on apples inoculated with Escherichia coli and Salmonella [10], on almonds inocu-
lated with Escherichia coli [11], and on grains and legumes infected with Aspergillus
spp. and Penicillium spp. [12]. Even if the results showed important reductions
according to the treatment time and used method, the above cited researches were
conducted only on previously inoculated samples and did not refer about possible
negative effects on the product quality traits such as changes in the peel colour and
chemical composition.
The present research aims to assess effects of non-thermal atmospheric air plasma
towards the indigenous microflora and the quality traits of fresh Abate Fetel pears,
a major pear cultivar in Italy. Possible negative effects on fruit quality traits were
analysed both immediately after the treatments and after storage for 5 days at 20C.
One of the two electrodes was covered by a 5 mm thick glass. The voltage at the
electrodes was produced by three high voltage transformers and power switching
transistors. The generated discharge was driven towards the fruits, placed at about
7 cm under the electrodes, by means of three fans mounted over the electrodes.
The discharge produced by the described configuration was electrically and
chemically characterized in a previous work [7]. Main results indicated that with an
input DC voltage of 19 V (chosen for the following microbiological and quality
tests) a potential difference of about 15 kV (peak-to-peak) was measured at the
electrodes. The plasma emission spectra showed the presence of very reactive spe-
cies such as the positive ion N2+ and NO and OH radicals. The emission of OH radi-
cals appeared to increase by increasing the humidity level of the air (RH).
The temperature profiles of the electrodes components (glass and brass) were
acquired for about 10 min by means of an infrared camera (FLIR, A325, FLIR
Systems) placed at about 40 cm from the discharge and operating in the range of
7.513.0 mm. The temperature of the electrodes components can indirectly give an
idea about the temperature reached by the gas plasma discharge generated between
the two parallel plates.
The Abate Fetel fruits were collected from the same farm immediately after har-
vest (Emilia Romagna region, Italy). The fruits were then stored at about 0C for
460 A. Berardinelli et al.
The efficacy of the device in reducing the superficial natural occurring microflora
was evaluated by exposing fruit samples to gas plasma for 10, 20, 30, 45, 60 and
90 min at RH of 60% (22C). For each treatment time five fruits were considered.
The plasma emission spectra at the cited atmospheric conditions were acquired
from 200 to 450 nm and at the distance of about 20 mm from the emission by using
a fibre optic probe (Avantes, FC-UV400-2) and a spectrometer (Avantes,
AVASpec-2048, resolution of 2.4 nm).
Following each treatment, pears were transferred into a sterile sampling bag
(International PBI S.p.A., Milan, Italy) and 100 ml of sterile saline solution (NaCl
0.9%) was added. Fruits were then hand-rubbed through the bag for 3 min to detach
the bacteria according to [13] and a 1-ml aliquot was used to prepare decimal serial
dilutions.
Enumeration of the surviving cells of total mesophilic bacteria, moulds and
yeasts was done by surface plating, in triplicate, 100 ml of the appropriate dilutions
onto Plate count Agar, Potato Dextrose Agar and agarized Sabouraud media with
added chloramphenicol (200 ppm), respectively. Plates were incubated at 30C for
48 h for bacteria and at 28C for 35 days for fungi.
The possible negative effects of the gas plasma on Abate Fetel pears quality traits
were assessed after 45 and 90 min of treatment at RH either of 23% and 60% and
22C (20 fruits/group). Immediately after the treatments possible weight losses and
changes occurring in the peel colour were quantified. The peel colour was measured
by means of a reflectance colorimeter in three different points of each fruit (Minolta
Chroma Meter CR-400, Minolta Italia S.p.A).
The peel colour was also measured after storage for 5 days at 20C with respect
to an untreated sample and together with the assessment of the Magness-Taylor
flesh firmness (MTf), the flesh soluble solids content (SSC) (Brix), and the antioxi-
dant capacity of peel and flesh (immediately under the peel). The MTf and SSC
measurements were carried out on opposite sides of each fruit by means of a manual
penetrometer (with 8 mm diameter probe) and a digital refractometer (PR-1, ATAGO
Co. Ltd.), respectively (only for the highest RH level). The antioxidant capacity was
assessed on the basis of the absorbance of the ABTS+ radical cation at 734 nm
(UV-Visible Spectrophotometer IE CARY, Varian) and the results were expressed in
35 Impact of Atmospheric Plasma Generated by a DBD Device 461
The analysis of the thermal profiles of the electrodes components acquired by means
of an IR camera during the discharge generation showed temperature values of the
brass and glass, which are in direct contact with the plasma, lower than 40C
(Fig. 35.2). It is reasonable to suppose that the discharge temperature is not too far
from this value. This low temperature is not surprising because of the presence of
the fans placed over of each pair of electrodes. In addition to directing the plasma
reactive species towards the product, the fans are also able to cool the electric
components.
The emission spectra of the discharge acquired at RH of 60% (22C) showed
dominant peaks of the N2 second positive system (l = 290440 nm) and the pres-
ence of the positive N2+ ion (l = 391 nm) (Fig. 35.3). Emissions of OH (l = 306 nm;
l = 280 nm; l = 285 nm;) and NO (l = 226248 nm) radicals were also observed.
The efficacy of the gas plasma treatments for superficial decontamination of Abate
Fetel pears has been evaluated towards the main microbial groups naturally occur-
ring on such fruit, i.e. total mesophilic aerobic bacteria, yeasts and moulds.
Data on viable cells detected immediately after exposure to gas plasma are
shown in Fig. 35.4. Comparison of all the microbiological data on treated fruits
evidenced that gas plasma treatments can effectively inactivate the fruit-associated
microflora although differences in resistance and dynamic were observed among
bacteria and fungi.
The relationship between the time of exposure to the plasma and the extent of the
reduction in viable cells was not linear regardless the microbial group. In general
yeasts and moulds seems to be more sensitive to the treatments and cell load
decreases of about 1 Log CFU/fruit were observed after a 1020 min treatment.
462 A. Berardinelli et al.
Fig. 35.2 Thermal profiles of the electrodes components after 10 min of treatment (A glass,
B brass)
70
N2 second positive system
60
50
Irradiance (W/cm2)
40
30
0
200 250 300 350 400 450
10 Wavelength (nm)
Fig. 35.3 Emission spectra of the plasma glow with a discharge voltage at electrodes of about
15 kV (RH = 60% at 22C)
The efficacy of the gas plasma generator increased by increasing the treatment time
and maximum reductions of 2.5 Log CFU/fruit were achieved both for yeasts and
moulds following the longest treatment.
On the contrary, the inactivation curve of total aerobic bacteria showed an initial
shoulder and no significant changes in viable cells were detected up to 30 min.
35 Impact of Atmospheric Plasma Generated by a DBD Device 463
7
Mesophiles
6 Yeasts
Moulds
Cell load (Log CFU/fruit)
0
0 10 20 30 45 60 90
Exposure time (min)
Fig. 35.4 Effect of exposure time on cell viability of total mesophilic aerobic bacteria, yeasts and
moulds on the surface of Abate Fetel pears
However, their population was reduced for longer treatments resulting in the same
maximum decontamination level as yeasts and moulds.
These results were obtained in the presence of humid air (RH = 60%, at 22C).
By using this RH level, Ragni et al. 2010 [7] evidenced that the decontamination
efficacy towards shell eggs is significantly influenced by the levels of OH radicals.
In particular, by means of the same afterglow device and voltage at electrodes, a
significant improvement of the decontamination power was observed for
S. Enteritidis by increasing the level of OH radicals obtained by modulating the
RH level from 35% to 65% (at 25C). In fact, maximum cell load reductions of
about 2.5 and 4.5 Log CFU/eggshell were obtained for RH levels of 35% and 65%,
respectively.
Since the measurements on fresh pears were conducted at high RH levels (60%
at 22C), the observed decontamination effect can be in part explained by the
presence of OH radicals which react with the fruit surface.
Guidelines for packing fresh or minimally-processed fruits and vegetables
generally specify a washing or sanitizing step to remove dirt, pesticide residues and
microorganisms responsible for quality loss and decay [16]. However, washing
procedures with water or chemical sanitizers typically result in only a 12 log
decrease in microbial counts [17]. Although chlorine is the most widely used disin-
fectant in industry to enhance the efficacy of the washing phase, recent outbreaks
associated with pathogen contamination in fresh-cut vegetables raised the concerns
about the efficacy of chlorine treatment in assuring the safety of the products [1].
Moreover, due to the environmental and health risks posed by the use of chlorine,
there is a trend in eliminating such chemical product in addition to the need for the
food industry to minimizing water consumption and wastewater discharge [18].
464 A. Berardinelli et al.
On the other hand during gas plasma treatments active species are present only
when the discharge is driven and disappear some milliseconds after the discharge
has been turned off. Such features assure that it is an almost harmless operation for
operators, consumers and materials, and some Authors reported that no toxic
residues remain on the sterilized foods [19].
The use of gas plasma for the decontamination of fruit pericarps from spoilage
and pathogenic microorganisms has already been proposed [9]. Although efficient
decontamination levels below the detection limit were achieved for S. cerevisae,
P. agglomerans and G. liquefaciens and E. coli deliberately inoculated onto the
pericarps of mango and melon, no study on the indigenous microflora contaminating
fruit has been performed. This aspect is quite important as natural contaminating
microflora can develop adaptation mechanisms which generally make it much more
resistant to decontamination procedures than the deliberately inoculated pathogens
or spoilage microorganisms.
No significant differences emerged between means for the peel colour and the mass
of the fruits analysed before and after the treatments (Table 35.1). It is likely that
differences in mass were not detectable due to the sample variance.
After a storage of 5 days at 20C, significant differences appeared for the peel
colour and the peel antioxidant capacity (Table 35.2). These differences were found
only after 90 min of treatment with respect to the untreated and 45 min treated samples.
L* parameter of fruits treated for 90 min at high RH level significantly decreased of
about 10% respect to the control, while the relative a* parameter significantly increased
of about 19% and 64% for low and high RH levels, respectively. The antioxidant
capacity of the peel appeared significantly lower (about 9%) in fruit samples treated
for 90 min both at low and high RH levels with respect to the untreated one. For the
flesh antioxidant capacity, MTf (N) and SSC (Brix) parameters (Table 35.3), no sig-
nificant differences between the tested samples were observed.
A possible reason for the observed pear darkening could be associated to
enzymatic activities and particularly to the polifenoloxidase (PPO), peroxidase and
phenylalanine ammonia-lyase (PAL) activities. In particular PAL is an important
plant enzyme that converts L-phenylalanine into trans-cinnamic acid, which in turn
is the precursor of various phenylpropanoids, such as lignins, flavonoids, and
coumarins. The phenolic compounds can then be oxidized by PPO producing brown
polymers that contribute to tissue browning in fruits. It is also reported that PAL
activity increases in response to several kinds of biotic and abiotic stresses including
wounding, exposure to ethylene, fungal infection [20].
Since 90 min treatments at both 23% and 60% RH levels (at 22C) appeared to
affect the peel colour and the peel antioxidant capacity, the increase in OH levels at
high RH values can not explain itself the oxidative processes occurring onto the fruit
surface during this kind of treatment. It is reasonable to suppose that also others
species detected in the emission spectra such as NO radicals can reach the fruit
surface and can play a role in the oxidative processes.
35 Impact of Atmospheric Plasma Generated by a DBD Device 465
Table 35.1 Influence of the gas plasma treatments on the quality of pear fruits immediately after
the treatments
Peel colour Mass
Sample L* a* b* g
45 min bt 61.1a(4.3) 2.8a(1.5) 41.2a(3.0) 199.8a(23.8)
RH = 23% at 59.7a(4.6) 3.7a(1.2) 40.7a(3.7) 199.7a(23.8)
a a a
45 min bt 63.0 (2.9) 2.5 (1.6) 42.1 (1.5) 218.8a(16.3)
RH = 60% at 61.5a(3.5) 3.3a(1.2) 42.0a(2.0) 218.6a(16.3)
90 min bt 61.5a(2.9) 3.7a(1.3) 42.3a(2.2) 222.7a(45.0)
RH = 23% at 61.0a(3.0) 4.1a(1.5) 42.5a(2.5) 222.4a(45.0)
a a a
90 min bt 60.7 (4.3) 6.7 (1.5) 41.4 (3.0) 206.6a(31.5)
RH = 60% at 60.1a(4.2) 6.6a(1.6) 42.1a(2.8) 206.3a(31.4)
a
Differences between means with the same exponent, before and after the same treatment, are not
significant at p-level value < 0.05 (bt before treatment, at after treatment). Standard deviations are
in brackets. The Peel colour was in terms of CIE L*a*b* colour space: L* lightness coordinate,
a* red coordinate, b* yellow coordinate. The fruit mass in measured in grams (g)
Table 35.2 Influence of the gas plasma treatments on the quality of pear fruits after storage for
5 days at 20C
Peel colour Antiox. capacity
Peel Flesh
Sample L* a* b* g Trolox/l g Trolox/l
a a a a
Control 63.0 (4.3) 5.8 (2.9) 41.2 (2.0) 0.76 (0.04) 0.67a(0.03)
a a a a
45 min RH = 23% 60.9 (4.2) 5.4 (4.0) 40.9 (3.4) 0.74 (0.03) **
45 min RH = 60% 61.3a(2.9) 5.9a(4.3) 41.6a(1.9) 0.76a(0.02) 0.68a(0.01)
90 min RH = 23% 60.1a(3.1) 6.9b(3.0) 42.1a(3.0) 0.69b(0.03) **
90 min RH = 60% 56.5b(3.1) 9.5c(3.9) 39.3a(5.3) 0.69b(0.03) 0.67a(0.01)
a,b,c
Differences between means with the same exponent within the column are not significant at
p-level value < 0.05 (for 3 g of peel and 5 g of flesh; ** not measured). Standard deviations are in
brackets. The Peel colour was expressed in terms of CIE L*a*b* colour space: L* lightness coordi-
nate, a* red coordinate, b* yellow coordinate. The antioxidant capacity was expressed in terms of
TROLOX (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid) equivalent antioxidant
capacity (grams of Trolox per liter)
35.4 Conclusions
The obtained experimental data showed that the DBD device used within this work
has a good potential in decontaminating Abate Fetel fruits. In fact, natural con-
taminating yeasts and moulds were reduced by 1 Log CFU/fruit after a 1020 min
exposure, while mesophilic bacteria resulted to be slightly more resistant to the
treatment. The decontamination efficacy of the gas plasma generator was enhanced
by using longer exposure treatments, and maximum reductions of 2.5 Log CFU/
fruit were achieved for yeasts, moulds and bacteria after 90 min. Despite the long
treatment times, no side effects on the quality traits such as peel colour, mass,
Magness-Taylor flesh firmness and flesh soluble solids content of the fruits were
detected immediately after the treatment and during storage. Such results are prob-
ably linked to the low temperatures reached during the treatment which makes such
a technology suitable for the decontamination of heat sensitive food products.
Although the temperature of the plasma was not be directly measured for the
unknown value of the emissivity of the produced discharge, the measurement of the
thermal profiles of the electrodes components proved that the temperature of both
the glass and brass does overcome 40C.
On the other hand, significant differences due to the gas plasma treatment were
detected for the pears treated for the longest time (90 min) in the presence of humid
air and then stored for 5 days. Even of the decontamination was achieved in after-
glow, the observed changes for the parameters representing the lightness (L*) and
redness (a*) and for the peel antioxidant capacity may suggest that the reactive spe-
cies produced in the gas plasma can interact with some enzymes, e.g. polifenoloxi-
dase, peroxidase and phenylalanine ammonia-lyase, or phenolic compounds and
vitamin C. On the other hand, it should be emphasised that pears peel can be easily
damaged due to chemico-physical, mechanical and thermal stresses.
Although the present study showed the potentialities of cold gas plasma as a
decontamination method alternative to fruit washing with chemicals (e.g. chlorine),
further studies are required in order to optimise the processing conditions to improve
the decontamination efficiency while reducing the side effects. In particular, the
working gas and distance of the products from the electrodes can be appropriately
chosen and modulated in order to avoid the losses observed for some superficial
quality traits after the longest exposures.
References
1. Seymour IJ (2003) Surface preservation for fruits and vegetables. In: Russell NJ, Gould GW
(eds) Food preservatives. Kluwer Academic/Plenum Publisher, New York, p 240
2. Allende A, Arts F (2003) UV-C radiation as a novel technique for keeping quality of fresh
processed Lollo Rosso lettuce. Food Res Int 36:739746
3. Palou L, Smilanick JL, Crisosto CH, Mansour MF (2001) Effect of gaseous ozone exposure on
the development of green and blue molds on cold stored citrus fruit. Plant Dis 85:632638
4. Moreau M, Orange N, Feuilloley MGJ (2008) Non-thermal plasma technologies: new tools for
bio-decontamination. Biotechnol Adv 26:610617
35 Impact of Atmospheric Plasma Generated by a DBD Device 467
36.1 Introduction
Plasma and electromagnetic fields treatments have been successfully applied over
the recent years for activation and decontamination of surfaces. Nowadays non-
thermal plasmas are considered functionally, energetically and ecologically as the
most efficient tool for pathogenic bacteria inactivation due to their high chemical
activity, short-time treatment and minimal materials destruction [1, 2].
Recently years these methods have also been successfully applied in agriculture
for improving the sowing quality of seeds. It has been shown in a number of previ-
ous studies that plasma and electromagnetic field pre-treatments of seeds stimulate
their germination and sprouting process, lead to suppression of fungal and bacterial
pathogens that cause various plant diseases [311]. At the same time the nature of
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 469
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3_36, Springer Science+Business Media B.V. 2012
470 I. Filatova et al.
36.2 Experiment
The principal scheme of the experimental set-up and the applied technique is shown
in Fig. 36.1. Tested species were processed with 5.28 MHz air plasma at a pressure
40 Pa. A capacitively coupled discharge was operated between two plane-parallel
water-cooled copper electrodes with a diameter of 120 mm placed in a stainless
steel vacuum chamber with the inner volume of 53.2 m3. The distance between
electrodes was varied between 20 and 40 mm according to the number and size of
the treated seeds. A supplied full specific RF power could be changed in a range
0.10.7 W/cm3 in dependence on treatment conditions.
A Petri dish with seeds to be treated by the plasma was put on the grounded
electrode before the vacuum chamber pumping. The exposure duration was 2, 5, 7,
10, 15 and 20 min. Under the experimental conditions the gas temperature did not
increase beyond 310C. Optical emission spectra were obtained with a Compact
Wide-Range Spectrometer S100 SOLAR LS in the optical range from 190 to
1,100 nm with average spectral resolution of 1 nm to identify the species present in
plasma during the treatment.
36 Fungicidal Effects of Plasma and Radio-Wave Pre-treatments on Seeds 471
Fig. 36.1 Experimental set-up: 1 alternator, 2 induction coil, 3 voltmeter, 4 vacuum chamber,
5 and 5 RF and grounded electrodes, 6 Petri dish, 7 quartz window, 8 lens, 9 monochromator,
10 camcorder, 11 personal computer
(seed vitality) was estimated after 3 days and germination laboratory test was
performed after 7 days. Each seed was considered to be germinated when it had
a radicle of 1 mm at least. The phytotoxicity test for control and treated seeds
was carried out after 7 days according to [13]. The STATISTICA 6.0 program
package was used for data statistical processing, inclusive of correlation and
regression analysis [14].
For analysis of seed health, the untreated and treated samples were incubated
on Potato Dextrose Agar (PDA) in Petri dishes at 23C to stimulate the develop-
ment of fungal colonies. The different fungi were then identified taking into
account their morphological and cultural characteristics according to [15]. The
analysis of seed-born infection of seedlings was carried out using the blotter rolls
method. The surface sterilized grains (kept for 3 min in 70% ethyl alcohol and
then washed in sterile water) were incubated in wet blotter rolls for 718 days
before the disease depth was estimated. Similarly, pathogenic diseases were
evaluated in the field conditions at the plant tillering stage. We tested 10 plant lots
(10 plants per plant lot) for each investigated culture during the field sanitary
inspections. The pieces of stems and leafs of infected plants with typical for each
fungal infection signs were taken for analysis throughout the plot area. Selected
samples were washed in sterile water, cut and plated in Petri dishes on PDA with
the cut side facing down on the nutrient medium. The infection was tested after
7 days of incubation.
The level of fungal infection P in the field tests was evaluated as the ratio between
the number of infected plants n and the total number N of investigated samples in
plant lot:
n
P= 100%. (36.1)
N
The disease severity R was estimated as follows:
( n b )
i
R= i
100%, (36.2)
N K
where ni is a number of infected plants with the same level of fungal lesion that
was corresponded to a mark b estimated in points (0 healthy, 1 weakly, 2
moderately and 3 deeply damaged), K is the highest mark of disease scale
(04).
A part of seeds were treated with appropriate fungicide vincit forte (manufac-
tured on the base of three reactants: flutriafol 37.5 g/l, thiabendazole 25 g/l,
imazalil 15 g/l) to study the effectiveness of plasma and radio-wave treatments in
comparison with common chemical methods of plant protection against fungal
infection.
36 Fungicidal Effects of Plasma and Radio-Wave Pre-treatments on Seeds 473
The laboratory and field germination characteristics of treated and control seeds of
blue lupin and field pea in dependence on exposure time are presented in Figs. 36.2
and 36.3 correspondingly. Significant correlations were found between the results
of laboratory and field tests. Both RF plasma and radio-wave pre-treatments did not
influence negatively on the sowing characteristics of seeds, but even improved their
germination within the optimal treatment conditions. The effect is less pronounced
in field tests that connected with influence of environmental conditions on germina-
tion and seed vitality during vegetation period.
The optimal exposure time was varied between 7 and 15 min in dependence on
sort of species differing in seed size, seed coat etc. Further increase of the treatment
duration (t > 20 min) did not lead to the positive effect and in some cases, for radio-
wave treatment led to germination decrease. Germination of seeds in field trials
after the electromagnetic field exposure was higher for optimal time than that in
control as follows: field pea by 13%, soy 8%, blue lupin 6% and barley 4%.
This effect for plasma treated seeds was lower (6% for field pea and 3% for blue
lupin) or statistically insignificant (for barley and wheat for example).
In spite of negligible enhancement of seed germination, a considerable increase
(up to 30%) of weight of 100 shoots was observed after the plasma treatment. It was
established that plasma treatment did not influence the length of the main root and
shoot of seedlings, but stimulated their branching especially on the 7th10th days of
ontogenesis (Fig. 36.4). Thus, the shoot mass accretion Dm for blue lupin at the
optimal exposure duration t = 7.5 min (Fig. 36.3a) amounted to 98% during the
period between the third and the 10th day for plasma treated seeds, while for control
samples it amounted to only 57%.
Therefore, the positive effect of plasma pre-treatment on seed germination
parameters becomes apparent not on the first days of ontogenesis but after a period
Fig. 36.3 Field germination and seed vitality of blue lupin (a) and field pea (b) as a result of
plasma and radio-wave seeds treatments
Fig. 36.4 The mean value of root/shoot length (a) and root/shoot mass (b) of sprouting seeds of
Lupinus angustifolius for control and plasma treated samples (20 seeds were tested in three repeti-
tions) in dependence on exposure duration measured on the 3rd, 7th and 10th days of ontogenesis
number and shoot weight) (Fig. 36.5). The best result was observed for 2.5-min
treatment. Moreover, sprouting seeds after the treatment developed better than the
controls and their vitality increased. The plant vitality that characterized the harvest
potency was evaluated as a ratio of number of ripe grown crops to the germinated
ones. It was established that radio-wave treatment of seeds increased the plant vital-
ity by 8% for wheat and barley owing to the larger number of productive stems per
unit area.
It was revealed that the efficiency of plasma and radio-wave seed treatments
depended on the degree of plant species domestication for agriculture with less
impact being achieved on the more domesticated species. In particular, the most
efficient effect of plasma and radio-wave treatments on seeds sowing characteristics
was observed for blue lupin and field pea as less domesticated cultures in compari-
son with wheat or barley.
The results of the laboratory tests have shown that seeds of blue lupin were infected
mainly with Fusarium and Alternaria, seeds of field pea with Fusarium, Alternaria
and Stemphilium, barley seeds with Fusarium. Both the plasma and radio-wave
treatments leaded to reduction of seeds infection with pathogenic fungi. The most
effective results for all tested species were observed after treatment for 10 and 15 min.
So, the field pea seeds exposure to plasma for 10 min resulted in decrease of Fusarium,
Alternaria and Stemphylium infections by 4%, 24% and 3% correspondingly
(Figs. 36.6 and 36.7). The Fusarium infection level in blue lupin seeds diminished by
9% after 15 min of plasma treatment. The longest exposure time was not effective in
seed enhancement because of influence on germination and seed vitality.
In the field conditions Anthracnose, Fusarium, Alternaria blackspot and
Stemphilium were tested as blue lupin diseases. Plasma and radio-wave seed
476 I. Filatova et al.
Fig. 36.6 The level of fungal infection of field pea (a) and blue lupin (b) seeds in dependence on
plasma treatment duration (laboratory test)
Fig. 36.7 The effect of plasma treatment on the level of fungal infection of field pea seeds (labora-
tory test)
pre-treatments during 1015 min leaded to decrease by 614% of the level of the
most harmful fungal infection (Fig. 36.8a). At the same time no fungicidal effect was
observed against Anthracnose. Plasma treatment for 515 min of soy seeds sup-
pressed by 4.87.2% Fusarium and Alternaria blackspot at early stages and advanced
growth stages (Fig. 36.8b). Similar results were observed for radio-wave treatment.
Seed pre-treatments for 1015 min were the most effective for both seed germination
enhancement and fungal infection reduction on sprouting seeds. Therefore, the fun-
gicidal effect for optimal conditions of seeds exposure to plasma and electromag-
netic field was comparable with the chemical fungicide pre-treatment of seeds. No
further decrease of the level of fungal contamination was observed in the field tests
for the treatment duration beyond 20 min practically for all tested species.
We suppose that the fungicidal effect of air plasma provides with the UV radia-
tion, seed surface bombardment with atomic oxygen and radicals formed in plasma
due to the oxygen contained molecules dissociation, and the formation of functional
groups on treated surface (Fig. 36.9) [13, 16].
36 Fungicidal Effects of Plasma and Radio-Wave Pre-treatments on Seeds 477
Fig. 36.8 Level of fungal infection of blue lupin (a) and soy (b) estimated in field trials as a result
of radio-wave (a) and plasma (b) treatments for different exposure durations
Fig. 36.9 Emission spectrum of RF air plasma recorded under conditions of plasma surface
decontamination
36.4 Conclusions
Acknowledgments This work has been supported partly by the State Committee on Science and
Technology of the Republic of Belarus (grant No. 11CP-015) and the Belarusian Republican
Foundation for Fundamental Research (grant No. T10-063).
References
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13. Naumova N (1970) Analyz semjan na gribnuju i bakterialnuju infekciju (The analysis of
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method: with the basics of statistical processing). Kolos, Moscow (In Russian)
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Subject Index
Z. Machala et al. (eds.), Plasma for Bio-Decontamination, Medicine and Food Security, 481
NATO Science for Peace and Security Series A: Chemistry and Biology,
DOI 10.1007/978-94-007-2852-3, Springer Science+Business Media B.V. 2012
482 Subject Index
Intracellular O
intracellular manipulation, 361 Octenidine, 281
intracellular parasites, 163 OH radical, 93, 201, 457
In vitro, 17, 121, 179, 321, 347, 361, 381, Optical emission spectroscopy, 45, 93, 121,
393, 403 149, 163, 201, 215, 231, 251, 265, 281,
In vivo, 17, 79, 251, 281, 321, 347, 361, 381, 311, 347, 445, 457
393, 403 Oxidative stress, 31
Irritative and imflammative potential, 321 Oxygen, 17, 107, 121, 179, 201, 347, 445
Ozone, 107, 445
K
Klebsiella peumoniae, 251 P
Pear fruit, 457
Penicillium crustosum, 57
L Peri-implantits, 191
Lactobacillus acidophilus, 215 Peroxynitrite, 67
Laser scanning microscopy, 281 Phototherapy, 251
Lesion-403 Pisum arvense, 469
Low pressure, 121, 469 Plaque forming unit, 79
Lupinus angustifolius, 469 Plasma
non-thermal plasma, 3, 17, 31, 45, 57, 67,
79, 93, 107, 121, 135, 149, 163, 179,
M 191, 201, 215, 281, 293, 301, 311, 321,
Medical 335, 347, 381, 417, 445, 457, 469
medical device, 3 plasma bullets, 149, 335, 381
medical therapy, 311, 361, 381, 393, 403 plasma chemistry, 67
Melanoma cells, 403 plasma cloud, 149
Mercury lamp, 231 plasma decontamination (sterilization),
Mesofilic bacteria, 457 3, 17, 31, 45, 57, 79, 93, 121, 135,
Microcathode sustained discharge, 107 149, 163, 179, 191, 201, 215, 231,
Microorganisms, 3, 17, 31, 45, 57, 67, 79, 93, 251, 265, 281, 301, 311, 361, 393,
121, 135, 149, 163, 179, 191, 201, 215, 417, 445, 457, 469
231, 251, 265, 281, 301, 311, 381, 393, plasma dentistry, 179, 191, 201, 215
417, 445, 457, 469 plasma diagnostics, 45, 93, 121, 149, 163,
Microplasma, 107 201, 215, 231, 251, 265, 281, 311, 335,
microplasma jet, 17, 45, 107, 201 347, 445, 457
Microwave plasma, 3, 121, 163 plasma disinfection, 179, 281, 301, 393
Microwave plasma jet, 3, 163, 311 plasma gun, 381
Mould, 57, 231, 457, 469 plasma inactivation, 17, 31, 67, 79, 93,
121, 135, 149, 163, 179, 215, 231, 281,
311, 417, 445, 457, 469
N plasma jet, 3, 135, 179, 191, 301, 321, 335,
Nanosecond pulsed electric fields, 361 381, 393
Neon, 381 plasma-liquid interaction, 67
Nitrate, 67 plasma medicine, 57, 281, 293, 311, 321,
Nitric oxide (NO), 107, 121, 293, 393, 445 335, 347, 381
NO therapy, 393 plasma micro jet, 17, 107, 201
Nitrite, 67 plasma splitting, 381
Nitrogen, 121, 135, 417 plasma torch, 347
Non-thermal plasma, 3, 17, 31, 45, 57, 67, 79, plasma-water interaction, 31, 67, 403
93, 107, 121, 135, 149, 163, 179, 191, Polihexanide, 321
201, 215, 281, 293, 301, 311, 321, 335, Post discharge, 93
347, 381, 417, 445, 457, 469 Psoriasis, 251
Nucleotide damage, 17 Protein damage, 17, 79
484 Subject Index
Pseudomonas aeruginosa, 135, 163, 251 Spore, 57, 231, 265, 445
Pulsed spore inactivation, 31, 45, 231
pulsed discharge, 31, 45 Sporulation, 57
pulsed electric field, 361, 403 Staphylococcus aureus, 3, 163, 251, 301,
393, 417
Stemphilium, 469
Q Sterilization, 3, 57, 79, 121, 231, 265
Quality traits, 457 Streptococcus
Streptococcus mutans, 31, 191, 215
Streptococcus sanguinis, 191
R Superoxide anion (O2-), 201, 293
Radical, 31, 93, 201, 281 Surface cleaning, 265
Radiofrequency Surgical mesh, 417
radiofrequency (RF) Synergy, 31, 67, 191, 231, 321
electromagnetic field, 469
radiofrequency (RF) plasma, 469
radiofrequency (RF) plasma jet, T
3, 135, 321 Teeth, 31, 179, 191, 201, 215
Raman microscopy, 281 Temperature measurement, 31, 45, 135, 149,
Reactive 163, 215, 281, 301, 321, 335, 445, 457
reactive nitrogen species (RNS), Tissue, 293, 335
17, 67, 293 tissue model, 293
reactive oxygen species (ROS), 17, 31, 67, tissue tolerable plasma (TTP), 281, 321
107, 293, 311, 347, 381 Titanium implant, 191
Remote exposure, 93, 231 Transepithelial electrical resistance
Risk assessment, 281 (TEER), 321
Root canal disinfection, 179 Transmission electron microscopy (TEM), 135
RNA damage, 17 Triticum aestivum, 469
Tumor treatment, 361, 381, 403
S
Saliva, 179, 191 U
Salmonella Ultraviolet
Salmonella enteritidis, 457 ultraviolet (UV) irradiation, 231, 281
Salmonella typhimurium, 31 ultraviolet (UV) radiation, 17, 163
Sarcina, 251 ultraviolet (UV) sterilization, 231, 251, 265
Scanning electron microscopy (SEM), 121, Underwater plasma, 403
179, 191, 201, 215
Schlieren photography, 403
Sealed package, 445 V
Seed Vacuum ultraviolet (VUV) radiation, 17, 163,
seed germination, 469 231, 251, 265
seed treatment, 469 Viable-but-non-culturable (VBNC), 135
Separation of plasma agents, 17, 31 Virulence test, 135
Shock waves, 403
Silver functionalization, 417
Singlet delta oxygen, 107, 201 W
Skin Water, 31, 57, 67, 201, 403
skin diseases, 251 water decontamination, 31, 265
skin disinfection, 281, 301, 393 Wound healing, 311, 321, 335, 347, 393
Spark, 31, 149, 293, 347
AC spark, 347
transient spark, 31 Y
Spectral sensitivity, 231 Yeast, 201, 457