International audienceThe transport and distribution phenomena of reactive oxygen and nitrogen species (RONS) into biological tissue following non-thermal plasma treatment have received much attention. The aim of this study is to test the efficacy of Plasma Gun (PG) on transporting the reactive species in an agar composed tissue phantom. RONS are generated on the agar gel by the plasma treatment and they continue to spread in the depth after plasma exposure. The amount of RONS after passing through the tissue phantom are strongly depending on discharge parameters such as distance to the target and capillary shape

Busco, Giovanni

Dozias, Sébastien

Grillon, C.

Omran, Azadeh,

Pouvesle, Jean-Michel

Robert, Eric

English

Archive Ouverte en Sciences de l'Information et de la Communication

HAL Id: hal-02263879
https://hal.archives-ouvertes.fr/hal-02263879
Submitted on 5 Aug 2019
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
Distribution and penetration of reactive oxygen and
nitrogen species through a tissue phantom after Plasma
Gun treatment
Azadeh Omran, Giovanni Busco, Sébastien Dozias, C. Grillon, Jean-Michel
Pouvesle, Eric Robert
To cite this version:
Azadeh Omran, Giovanni Busco, Sébastien Dozias, C. Grillon, Jean-Michel Pouvesle, et al.. Distribu-
tion and penetration of reactive oxygen and nitrogen species through a tissue phantom after Plasma
Gun treatment. 24th International Symposium on Plasma Chemistry (ISPC24)„ Jun 2019, Naples,
Italy. ￿hal-02263879￿
Distribution and penetration of reactive oxygen and nitrogen species through a 
tissue phantom after Plasma Gun treatment 
 
A. Valinataj Omran1, G. Busco2, S. Dozias1, C. Grillon2, J-M Pouvesle1 and E. Robert1 
 
1 GREMI UMR 7344 CNRS/Université d’Orléans, 45067 Orléans, France 
2 Centre de Biophysique Moleculaire, UPR 4301CNRS rue Charles Sadron, 45071 Orléans, France 
 
Abstract: The transport and distribution phenomena of reactive oxygen and nitrogen species 
(RONS) into biological tissue following non-thermal plasma treatment have received much 
attention. The aim of this study is to test the efficacy of Plasma Gun (PG) on transporting the 
reactive species in an agar composed tissue phantom. RONS are generated on the agar gel by 
the plasma treatment and they continue to spread in the depth after plasma exposure. The 
amount of RONS after passing through the tissue phantom are strongly depending on 
discharge parameters such as distance to the target and capillary shape. 
 
Keywords: Plasma Gun, RONS, tissue phantom, target gap, nozzle shape. 
 
1. Introduction 
Atmospheric pressure plasma jets have attracted a 
considerable attention for numerous applications in 
medicine and biology. The biomedical effects of plasma 
are strongly linked to the plasma generated RONS. An 
important issue in application of cold atmospheric plasma 
for tissue treatment concerned the penetration of RONS 
into biological tissue [1]. Because of the unique properties 
(soft and hydrated nature), RONS distribution and 
penetration can vary from tissue to tissue. While human 
skin is able to resist the penetration of many molecules, 
smaller molecules and species can pass through the corneal 
layer of the epidermis [2]. 
The tissue properties such as roughness and conductivity 
can influence the plasma treatment. Also, the gap distance 
(i.e., distance between the tip of the capillary and the 
target) may lead to variation of the plasma characteristics 
such as concentration of reactive species and gas 
temperature which can in turn change the effectiveness, 
distribution and penetration of the plasma on/in the tissue.  
  In the present work, we have focused on the RONS 
transportation and distribution in an agar tissue phantom 
after PG treatment. This agar model is very significantly 
different from real biological tissue but it can allow to get 
some precious information on transport of RONS in water 
containing media. Our results clearly and visually indicate 
that RONS are accumulated in the tissue phantom after the 
plasma treatment, and that continue to diffuse over and 
across the tissue. The influence of different nozzle shapes 
and gap distances on the distribution and penetration of 
RONS is studied. 
 
2. Material and Method 
Figure 1 shows a schematic of the experimental setup 
and two different nozzles. The plasma studied in this work 
is Plasma Gun (PG) composed of a coaxial dielectric 
barrier discharge reactor (Fig. 1) [3]. A 2 cm long powered 
electrode is set inside the 12 cm Pyrex capillary, 4 mm 
inner diameter and 6 mm outer diameter. In straight tube 
(Fig. 1 (a)), the inner diameter of the nozzle is 4 mm but in 
a tapered one (Fig. 1 (b)), the inner diameter at the end of 
the tube is 1.5 mm. 
 The helium flow rate through the reactor capillary is 0.5 
slm. In this work, the PG device is driven by μs duration 
voltage pulses (positive polarity, repeated at 2 kHz). The 
applied voltage is measured using a high-voltage probe 
(Tektronix P6015A) and the discharge current on the agar 
target is measured using a 100 Ω shunt resistor. Also, 
discharge power is measured from V-Q Lissajous figures 
obtained using a 1.5 nF capacitor connected to the 
grounded electrode in series. 
 
 
 
Fig. 1. Schematic diagram of the experimental set-up for a) 
straight nozzle and b) tapered nozzle c) RONS delivery 
through agar gel. 
 
Figure 1 (c) shows the setup which can allow to visualize 
the penetration of RONS through the tissue phantom. A 
tissue phantom, made of a 2 mm thick agar gel, is placed 
on top of a 96-well plate; each well is filled with phosphate 
buffered saline (PBS, pH 7.4). The plasma plume produced 
by the cold atmospheric PG contacts the top of the agar 
surface. Then, the concentrations of RONS generated in 
PBS are measured. The experiments are repeated at least 
three times for each condition. 
Preparation of agar target 
  The agar target is prepared by dissolving 1.5% agar 
powder in physiological saline solution. The solution is 
heated on a conventional heating magnetic stirrer until all 
the powder is dissolved. Agar gels of 2 mm thickness are 
prepared by pouring 4,4 mL of solution into an 53 mm 
diameter polystyrene Petri dish. The agar is stored at 4ºC 
for 12 hours before use. Square sections of approximately 
15 x 15 mm were cut using a scalpel and removed with a 
lab spatula. These agar squares, used for RONS penetration 
experiments, were laid on the wells of a 96 well-plate 
overfilled with the recipient medium. 
In addition, the agar gel with KI-starch is used to evaluate 
the spatial distribution of oxidation reactions induced by 
PG. The agar target is prepared by dissolving 1.5% agar 
powder, 0.3% potassium iodide and 0.5% starch 
((C6H10O5) n) in physiological saline solution. The KI-
starch gel reagent can detect hydroxyl radicals (OH), 
atomic oxygen (O), O3, H2O2, and HO2 radicals. 
 
H2O2 measurement in PBS solution 
  To measure the concentration of hydrogen peroxide 
passed through the gel into PBS after PG treatment, the 
Amplex™ Red assay kit was used. The Amplex red 
molecule is converted in the highly fluorescent resorufin in 
presence of H2O2 and peroxidase (HRP). For fluorescence 
intensity measurement, we used a microplate reader 
spectrofluorometer (Victor 3V, PerkinElmer), excitation 
wavelength at 531/25 nm and emission wavelength of  
605/10 nm .  
  Not treated (NT) and helium treated gels were used as 
controls. Fifteen minutes after each treatment, the agar gel 
was removed and 50 µl of PBS containing diffused ROS 
were collected and transferred into individual wells of a 96-
well plate. The working solution of 100-μM Amplex red 
reagent and 0.2-U/mL HRP were prepared, following the 
manufacturer’s protocol. Then, 50 μL of the working 
solution was mixed with the same volume of the loaded  
sample. The 96-well plate was incubated in the dark at 
room temperature for 30 min and then read with the 
microplate reader. 
 
RONS measurement in PBS solution: 
  The RONS concentrations passing from the agar gel 
target to the PBS are measured using Dihydrohodamine 
123 (DHR, Thermo Fisher Scientific). DHR is a non-
selective reactant, reacting with H2O2 and peroxynitrite 
(ONOO-). It may react with other derivatives of molecular 
oxygen, including 1O2 . RONS are detected by the 
formation of fluorescent Rhodamine123 after the reaction 
with nonfluorescent DHR. In this experiment, we used 
DHR 123 5µM in PBS. The solution (420 µl) was 
distributed in each well of a 96 well plate and the 
15mm15mm agar gel targets with 2 mm thickness were 
placed over the top of the well so that the film contacted 
the PBS inside the well. Fifteen minutes after the treatment, 
the agar gel was removed and 200µl of the PBS containing 
the fluorescent probe were collected and transferred in a 
new 96 well plate. Fluorescence was measured using the 
microplate reader spectrofluorometer, excitation at 485nm 
and emission at 535nm. 
 
3. Results 
  The waveforms of applied voltage and current of the 
plasma jet are presented in Fig. 2(a); it can be found that 
the peak applied voltage is 9 kV and the frequency is 2 kHz.  
Since the discharge power is applied in several short 
current pulses it is not a trivial task to calculate the power 
dissipated in the discharge. The method commonly 
employed for obtaining the discharge power is based on the 
charge–voltage Lissajous figure. The area of the closed Q-
V loop for one period of the applied voltage is equal to the 
energy dissipated per one cycle. The mean discharge power 
is then simply the energy per cycle multiplied by the 
working frequency. A Q–V Lissajous figure of the plasma 
jet is shown in Fig. 2 (b) for tapered tube. It is obtained for 
applied voltage amplitude of 9 kV, 2 kHz signal frequency, 
gas flow of 0.5 slm and 10 mm, 15 mm and 20 mm distance 
between the tapered nozzle and the substrate. At these 
operating conditions, the area of the Lissajous figure for 10 
mm, 15 mm and 20 mm gaps shown in Fig. 2 are 45 µJ, 32 
µJ and 25 µJ giving a power of 90 mW, 64 mW and 50 
mW, respectively. As expected, the amount of charge 
transferred to the agar target during a discharge cycle 
markedly decreased with increasing target gap. In addition, 
about the same value have been measured for straight tube. 
 
Fig. 2. a) Voltage pulse applied on the electrode and current 
time evolution measured across the target with a 100 Ω 
resistor for 10 mm target gap b) Q-V plots measured at 
different target gaps for tapered tube. 
 
  We used a KI-starch gel to show the oxidation reactions 
on the tissue surface [4]. As shown in Fig. 3, the color of 
the surface of the KI-starch gel changed from colorless to 
purple after plasma treatment. As expected, for different 
plasma conditions, an area with a strong purple color is 
observed in the center of the dish, which indicates that 
contact of the plasma with the tissue surface induces 
oxidation reactions caused by the generated ROS. 
  Images of the treated KI-starch gel dish were exported 
and processed in ImageJ (Fig. 4).  Real dimension of the 8-
Bit grey scale images were assigned in mm units using the 
set scale function. The intensity and the width of the purple 
spot were measured by tracing a straight line, passing 
through the center of the treated area. Data from each plot 
profile was exported in Origin software for analysis. 
  Concerning the plasma/gap distance (Fig. 3 and 4), the 
central strong purple area exhibits a bigger radial 
expansion and a decrease in intensity when the gap is 
increased from 10 to 20mm. At small gap, the central point 
on the agar gel surface shows a less pronounced purple 
color. In a first approximation, we may think that this could 
be due to the hollow shape distribution of the ROS in the 
plasma plume [5], but this is not totally consistent with the 
diffusion of the ROS within the agar medium elsewhere. 
Additional study must be conducted to study the potential 
effect of ablated material in that area. Figures 3 and 4 also 
show the strong influence of the nozzle shape on the ROS 
distribution. 
 
Fig. 3. Photographs of the KI-starch gel after plasma-jet 
treatment for 120 s with different nozzles and plasma-
treatment distances. 
 
Fig. 4. Relative comparison of ROS concentration 
distributions on the KI-starch gel with different plasma 
treatment distances for a) straight nozzle and b) tapered 
nozzle. 
 
  The concentration of the reactive species that penetrate 
into the agar gel is of particular interest for a better 
understanding of the transport of RONS in the biological 
tissue. The concentration of H2O2 and RONS in PBS after 
penetration through the agar film under the different gaps 
and different nozzles are shown in Fig. 5. It is found that 
the concentration of H2O2 and RONS decreases with the 
increase in the gap to 20 mm. The treatment distance 
affects the energy deposition on the target and also the 
concentration of the RONS in the PBS. However, the 
change in concentrations of RONS is not in proportion to 
the energy deposition. It seems that the contribution of the 
humidity in production of the RONS is more than the 
charge deposition. A closer treatment distances result in a 
greater level of evaporation creating a more humid 
atmosphere that may affect the RONS concentration. In 
addition, significant higher concentrations are obtained for 
plasma treatment with tapered nozzle than the case of 
straight nozzle. In tapered tube, the He gas flow produces 
a more localized point of contact with the agar film, 
together with a higher gas velocity, which might result in a 
localized increase in RONS density at the gas-surface 
interface that may further facilitate the transport of RONS 
into the agar film. Such enhanced RONS penetration was 
measured to be more pronounced using DHR probe than 
with the H2O2 selective Amplex red assay. 
 
 
 
 
Fig. 5. The concentrations of (a) H2O2 and (b) RONS of the 
treated PBS samples after penetration through the agar gel 
under the different target gaps for tapered and straight 
tubes. Vp: 9 kV, f: 2 kHz, He flow: 0.5 slm. 
 
 
4. Conclusions 
The knowledge of plasma-tissue interaction is important 
for the development of cold plasma technology for biology, 
medicine and food industry. In this work, we used a tissue 
phantom using an agar gel that acts as a physical target and 
barrier to investigate how the RONS produced by a PG can 
0
1
2
3
4
5
6
7
8
9
10
10 mm 15 mm 20 mm
H
2O
2 
(µ
M
)
Tapered
Straight
a)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
10 mm 15 mm 20 mm
C
o
n
ce
n
tr
at
io
n
 o
f 
R
1
2
3
 (
a.
 u
.)
Tapered
Straight
b)
penetrate through the tissue. The concentration of RONS 
produced by a PG distributing on the surface and 
penetrating through the agar in different nozzle to target 
distance and nozzle shape were measured. As previously 
shown by other teams, we confirm that RONS have the 
ability to diffuse through few millimeter thickness of water 
containing medium. The produced RONS concentration is 
strongly linked to the application conditions such as 
distance to the target and shape of the plasma applicator 
inducing different conditions of air mixing with the helium 
flow. This emphasize on the fact that diagnostics of plasma 
plume and long lived species production must be 
performed in the closest condition to the real biological 
ones. 
These results shows the potential of water containing 
tissue phantoms in determining an illustrative basis for 
specifying optimal and/or safe plasma conditions and 
provide consumer confidence in utilizing cold atmospheric 
plasma in medicine and cosmetics. 
 
 
5. Acknowledgments 
  This work was supported by Cosmetosciences, a global 
training and research program dedicated to the cosmetic 
industry, located in the heart of the Cosmetic Valley, this 
program led by University of Orléans is funded by the 
Région Centre-Val de Loire, France 
 
 
6. References 
[1] E. J. Szili, J. W. Bradley, R. D. Short, Journal of 
Physics D: Applied Physics, 47, 152002 (2014). 
[2] J. D. Bos, M. M. Meinardi, Viewpoint, 9, 165 (2000). 
[3] E. Robert, V. Sarron, D. Riès, S. Dozias, M. 
Vandamme, J. M. Pouvesle, Plasma Sources Science and 
Technology, 21,034017 (2012). 
[4] T. Kawasaki, K. Kawano, H. Mizoguchi, Y. Yano, K. 
Yamashita, M. Sakai, Int. J. Plasma Environ. Sci. 
Technol., 10, 41 (2016). 
[5] N. Bourdet, M. Laroussi, A. Begum, and E. Karakas, 
Journal of Physics D: Applied Physics,42, 055207 (2009). 
 
 
 
 


Distribution and penetration of reactive oxygen and nitrogen species through a tissue phantom after Plasma Gun treatment

Omran, Azadeh, Valinataj

HAL Université de Tours

https://hal.archives-ouvertes.fr/hal-02263879/document

Distribution and penetration of reactive oxygen and nitrogen species through a tissue phantom after Plasma Gun treatment

Abstract

Similar works

Full text

Available Versions

Archive Ouverte en Sciences de l'Information et de la Communication

HAL Université de Tours