Ahmed Chebbi, Claire Staunton, Vic Law, and Denis Dowling

1. Variable Frequency Atmospheric Plasma Source for
Skin Treatment Applications
Ahmed Chebbi, Claire Staunton, Vic Law, and Denis Dowling
University College Dublin

2. What is Plasma?
• Defined as the fourth state of matter.
• When adding energy (DC, microwave,
radiofrequency) to a gas, the electrons
separate from the nucleus and move around
freely producing ionized gas (plasma).
• 99% of all matter in the visible universe is in
the state of plasma.
• Atmospheric plasma has been used
extensively in industry for surface
treatment, activation and modification.

3. Plasma Medicine
• Plasma medicine is the intersection of plasma science
and technology with biology and medicine for:
– Plasma based sterilization
(e.g. for medical devices)
– Direct therapeutic plasma
applications (e.g. for
wound healing)
– Plasma modification of
biomedical surfaces (e.g.
for hip implants)
•Cancer treatment
•Blood coagulation
•Dentistry
•Orthopedics
•Wound healing
•Skin disease

4. Plasma Medicine for Wound Healing
• What’s in plasma? UV, heat, Reactive Oxygen
Species (ROS), Reactive Nitrogen Species (RNS)…
• The plasma wound healing effect is obtained
through a reduction of the bacterial load count
on the wound surface, and in some case through
the enhancement of critical phases in the wound
healing cascade.
• Reactive oxygen and nitrogen species produced
by the plasma are mainly responsible for this
bactericidal effect.
• Plasma processing parameters affect the
production of reactive species.
• An optimal treatment regime needs to be
identified in order to obtain the highest
bactericidal effect.
A 61-year-old patient with venous
ulcers: wounds before plasma
treatment (a), after 7 (b) and after
11 treatments (c). With a daily
plasma therapy (MicroPlaster®) of 2
min. At the beginning of plasma
treatment Klebsiella oxytoca and
Enterobacter cloacae were
detectable, after 11th treatment (23
days later) swabs were sterile*.
* Plasma Medicine: Possible Applications in Medicine, Journal of German Society of
Dermatology, 2010-8

5. Volumedischarge
20 kV
power supply
Teflon tube
(72 mm x 15 mm)
Work surface
Gap
distance
Needle
electrodes
HDPE
HV Probe
Plasma
plume
Atmospheric Plasma Jet at University College Dublin
Variable
frequency
power
Quartz tube
Glass
Processing parameters:
Voltage: 0-300 V
Frequency: 0-500 kHz
Helium Flow rate: 0-20 l/min
Helium Gas
• Study objectives:
• Using a novel variable frequency plasma source, to identify an adequate
plasma processing window for the treatment of skin (wound healing
applications) without causing damage,.
• To demonstrate the effect of plasma treatment on bacterial cells.

6. Optimal Plasma Treatment Regime
• Optical Emission Spectroscopy (OES) is a
qualitative elemental analysis used to detect
chemical species in the plasma.
0
5000
10000
15000
20000
25000
0 50 100 150 200 250 300 350 400 450 500
Intensity(a.u.)
Frequency kHz
OH NO2 O He
OES
Probe
• The frequency of the plasma power supply was varied from 0 to 500
kHz in order to identify the treatment regime which yields the highest
production of active species.
• The highest production of NO and OH was found at a frequency of 160
kHz (with 100 V and 10 l/min of helium flow rate).
160 kHz

7. Effect of Plasma Treatment on E.Coli
1
10
100
1000
Control 140 kHz 160 kHz 180 kHz
Plasma for 2 Minutes at 140, 160, and 180 kHz
CFU1013/ml
1
10
100
1000
10000
100000
1000000
Untreated 2 min 4 min 6 min
CFU/mlx106
Plasma at 160 kHz for 2, 4, 6 minutes
140 kHz 180 kHz160 kHzControl Untreated 2 min 4 min 6 min
The highest production of active species
resulted in the highest reduction of
bacterial load count in vitro.
Longer plasma exposure results in higher
reduction of bacterial load count ex vivo on pig
skin samples.
In order to correlate the production intensity of
plasma species with the bactericidal effect:
different frequencies were used to treat
solutions containing the same amount of E.Coli.
After plasma treatment and serial dilution, 100
µl was spread on agar plates and left overnight.
In vitro Ex vivo
An ex vivo pig skin model was used to investigate the
effect of plasma exposure time on the bactericidal
effect. Fresh pig skin sample were used and a known
amount of bacteria was placed on the surface. After
plasma treatment, skin samples were PBS washed and
a 100µl dilution spread on agar plates.

8. Sensitivity of Wound Pathogens to Plasma Treatment
Order of susceptibility to atmospheric plasma treatments
Klebsielle (G-) > E. coli (G-) > P. Aeruginosa (G-) > S. Aureus (G+) > B. Subtilis (G+)
• A comparison was made on the sensitivity of both gram negative and gram positive bacteria to the
plasma treatment under the same processing conditions.
• Gram negative bacteria were found to be far more susceptible to the treatments (e.g. E.coli (Gram
negative) and B. subtillis (Gram positive)).
• The relative lack of structural damage observed for Gram-positive bacteria is due to their thicker murein
layer, making them more rigid and thus increasing their tensile strength.

9. Flow Cytometry Analysis
L) Untreated control sample, no dye uptake (healthy cells) R) 2 min plasma treatment: 3 populations
corresponding to healthy cells, depolarised membrane (BOX) and permeabilised membrane (PI, full cell death)
• The bacterial cell (E.coli) is exposed to a fluorescent dye, which is adsorbed only when damage occurs. Two
flourescent dyes were used during this study: Bisoxonol (BOX) for the detection of a depolarised membrane and
Propidium Iodide (PI) for the detection of a fully permeabilised membrane.
• With increasing plasma intensity the progression towards cell death was clearly evident. The mechanism observed
was initially a decrease in cell membrane potential culminating in full membrane permeabilisation as shown by the
initial uptake of the dye BOX, followed by the uptake of PI.

10. Conclusions
 We demonstrated an antibacterial activity of the variable
frequency helium plasma system in vitro (E coli solution)
and ex vivo (pig skin).
 We optimized the plasma treatment for maximal bacterial
load reduction while minimising damage to pig skin.
 A once-off plasma treatment for 120 seconds led to a 1 log
reduction of bacterial count load in vitro and on pig skin
samples inoculated with E.coli.
 Higher treatment times of up to 6 minutes led to a 4 log
reduction in bacterial count load ex vivo (on pig skin).
 Gram negative bacteria were more susceptible to plasma
exposure than gram positive bacteria.
 Flow cytometry data showed that the cell breakdown
pathway consists of membrane depolarisation and
eventual permeabilisation.