International audienceCold Atmospheric Plasmas (CAPs) have been shown to lower the pH of the treated target. CAP-induced acidification can have beneficial effects on the treated tissue (e.g. disinfection and healing). Conversely a tissue hyper acidification can lead to permanent damages with dramatic consequences. Before starting in vivo plasma treatments, preliminary tests should be carried in order to tune the acidifying effect induced by CAP exposition. Here we propose a tissue model that can be a useful tool to visualize and measure pH changes induced by plasma treatment

Busco, Giovanni

Grillon, Catherine

Pouvesle, Jean-Michel

Ridou, Loick

Robert, Eric

Valinataj Omran, Azadeh

English

HAL Université de Tours

Fluorescein-agarose gel as a tissue model to visualize and measure local CAP-induced acidification

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Fluorescein-agarose gel as a tissue model to visualize
and measure local CAP-induced acidification
Giovanni Busco, Azadeh Valinataj Omran, Loick Ridou, Jean-Michel
Pouvesle, Eric Robert, Catherine Grillon
To cite this version:
Giovanni Busco, Azadeh Valinataj Omran, Loick Ridou, Jean-Michel Pouvesle, Eric Robert, et al..
Fluorescein-agarose gel as a tissue model to visualize and measure local CAP-induced acidification.
24th International Symposium on Plasma Chemistry (ISPC24), Jun 2019, Naples, Italy. ￿hal-02263357￿
Fluorescein-agarose gel as a tissue model to visualize and measure local 
CAP-induced acidification 
 
 
Giovanni Busco1, Azadeh Valinataj Omran2, Loïck Ridou1, Jean-Michel Pouvesle2, Eric Robert2 and Catherine 
Grillon1 
 
1Centre de Biophysique Moléculaire, UPR4301CNRS, 45071 Orléans, France 
2GREMI, UMR 7344 CNRS/University of Orléans, 45067 Orléans, France 
 
 
Abstract: Cold Atmospheric Plasmas (CAPs) have been shown to lower the pH of the 
treated target. CAP-induced acidification can have beneficial effects on the treated tissue 
(e.g. disinfection and healing). Conversely a tissue hyper acidification can lead to 
permanent damages with dramatic consequences. Before starting in vivo plasma 
treatments, preliminary tests should be carried in order to tune the acidifying effect 
induced by CAP exposition. Here we propose a tissue model that can be a useful tool to 
visualize and measure pH changes induced by plasma treatment.  
   Keywords: agarose gel model, pH measurement, fluorescein, plasma jet 
 
 
1. Introduction 
Once in contact with the atmosphere and water, 
Cold Atmospheric Plasmas (CAPs) initiate complex 
chemical reactions that lead to Reactive Oxygen and 
Nitrogen Species (RONS) generation. The produced 
RONS are mostly responsible for the biological effects 
observed during and after CAP treatment. Besides their 
oxidative effect, some CAP-generated species possess 
also an acidic character. The acidifying effect induced 
by CAP on treated liquids is widely known [1, 2].  
While measuring the pH drop in liquids is relatively 
easy, the evaluation of the pH on solid surface is more 
complex. Flat pH-meters permit today to measure the 
pH on solid surfaces and organs like the skin. However, 
once applied to the solid target, these flat pH-meters 
give an average value of the pH on the covered surface.  
The relatively large surface of the pH-meter electrode 
does not permit to appreciate pH distribution, 
especially during a focalized CAP treatment where 
RONS are concentrated on a small surface. In this work 
we took advantage of the fluorescein fluorophore 
whose fluorescence is pH sensitive [3-5] to study the 
CAP induced acidification in agarose tissue models.  
2. Method 
In this work we used a modified protocol of our 
recently published method [6]. Briefly a buffered 
physiological solution (130mM NaCl, 5mM KCl, 2mM 
MgCl2, 5.5mM HEPES) was prepared at 6 different pH 
ranging from 7.4 to 2.5. Fluorescein sodium salt  
 
(50µM) and DNA grade agarose (0.02g/ml) were added 
to each solution and then agarose was gently melted in 
a microwave.  Gels at different pH were poured in 6-
well plates in order to obtain a 2 mm thick gel/well. 
Gels were let to solidify and then kept at 4°C. Before 
plasma treatment plates were allowed to equilibrate at 
room temperature. Gel fluorescence was then imaged 
using the Typhoon™ FLA 9500 biomolecular imager 
(GE Healthcare Life Science) using an excitation laser 
at 473 nm and a Y520 emission filter with a wavelength 
range ⩾ 520 nm. Immediately after the scan, the 6-well 
plate was exposed to CAP treatments. We treated the 
gel at initial pH of 7.4 (as generic organ model) and the 
gel at initial pH of 5.5 (as skin model). Plasma 
treatment was performed using a Plasma Gun [7] with 
a negative polarity high voltage pulses having a peak 
amplitude of −14 kV, full width at half maximum of 2 
μs duration. Gels were exposed for 5 min to the thin jet 
produced at the tip of a thin tapered capillary (1.5 mm 
internal diameter). Helium was used as feeding gas at a 
flow rate of 0.5 SLM. After the treatment, the plate was 
scanned again to image the changes in the fluorescence 
induced by CAP exposure. 8-bit grey scale images were 
imported in ImageJ for data analysis. The scan of the 6-
well plate before CAP treatment was used to calibrate 
the fluorescence signal vs the 6 known pH. A 
calibration curve was thus obtained and used to 
measure the CAP-induced pH drop. To better visualize 
the pH variations, grey scale images were converted in 
spectrum pseudocolor in ImageJ. pH analysis on the 
plasma treated surface was done by tracing a straight 
line passing by the middle of the acidified area and 
measured values were plotted in a graph (figure 2). 
 
3. Results 
 
Immediately after the CAP treatment, acidified area 
appears as a darker spot on the green agarose gel. 
Fluorescein fluorescence is gradually quenched when 
the pH in the medium decreases. To better visualize the 
CAP-induced acidification, the 8-bit grey scale images 
were converted in pseudocolors (figure 1). The spatial 
pH distribution, measured by tracing a straight line 
passing through the CAP-exposed surface showed a pH 
decrease of one unit in the generic organ model, the 
generic organ model and a slightly higher decrease in 
the skin model (figure 2).  The lower acidification 
observed in the skin model could be attributed to the 
HEPES buffering activity having its optimum in the pH 
range between 8.2 and 6.8. 
 
 
 
 
 
Figure 1. Visualization of the CAP-induced 
acidification on the fluorescent agarose gel models. To 
better visualize the acidified area, 8-bit images were 
converted in spectrum pseudocolor in ImageJ 
 
4. Conclusions 
In this work we propose a novel technique useful to 
evaluate and measure the CAP-induced acidification on 
agarose-gel tissue models. Agarose-gels with initial pH 
of 7.4 or 5.5, mimicking respectively a generic organ or 
the outer skin pH, were used to evaluate the acidifying 
effect of plasma treatment. These models can be useful 
during CAP treatment planning, especially when a 
focalized treatment should be administered. 
Controlling the local induced acidification can help to 
avoid hyper-acidification that can cause chemical burn 
on the CAP exposed tissue. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. Plotted graphs of pH measured along a 
manually traced straight line passing through the 
CAP-exposed surface 
 
 
5. Aknowledgement 
 
   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. Giovanni Busco, Azadeh Valinataj Omran and 
Loïck Ridou are supported by Cosmetosciences ( 
PLASMACOSM Project, Grant 2015-00103497 and 
PLASMACOSM-2 Project, Grant 2017-00118114
after CAP treatment before CAP treatment 
generic organ model 
before CAP treatment after CAP treatment 
skin model 
2.0
2.5
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3.5
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4.5
5.0
5.5
6.0
6.5
7.0
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8.0
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H
distance (mm)
2.0
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distance (mm)
before CAP treatment after CAP treatment
skin model 
generic organ model 
6. References 
 
[1] Brisset J.-L., et al., Acidity control of plasma-
chemical oxidation: applications to dye removal, 
urban waste abatement and microbial inactivation. 
Plasma Sources Sci. Technol. 20 034021 (2011). 
[2] Oehmigen, K., et al., The Role of Acidification for 
Antimicrobial Activity of Atmospheric Pressure 
Plasma in Liquids. Plasma Processes and 
Polymers, 7(3‐ 4): p. 250-257. (2010) 
[3] Emmart, E.W., Observations on the absorption 
spectra of fluorescein, fluorescein derivatives and 
conjugates. Archives of Biochemistry and 
Biophysics, 73(1): p. 1-8. (1958) 
[4] Leonhardt, H., L. Gordon, and R. Livingston, Acid-
base equilibriums of fluorescein and 2',7'-
dichlorofluorescein in their ground and fluorescent 
states. The Journal of Physical Chemistry, 75(2): p. 
245-249. (1971) 
[5] Martin, H., et al., Sodium fluorescein as a retinal pH 
indicator? Physiological Measurement, 26(4): p. 
N9. (2005) 
[6] Busco G., et al, Cold atmospheric plasma-induced 
acidification of tissue surface: visualization and 
quantification using agarose gel models.  J. Phys. 
D: Appl. Phys. 52 24LT01 (2019) 
[7] Robert, E., et al., Characterization of pulsed 
atmospheric-pressure plasma streams (PAPS) 
generated by a plasma gun. Plasma Sources Science 
and Technology, 2012. 21(3): p. 034017. 
 
 
 
 
 
 
 
 
 


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Fluorescein-agarose gel as a tissue model to visualize and measure local CAP-induced acidification

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