22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Preliminary study of low pressure low temperature RF plasma confined inside
sterilization pouches for the sterilization of medical devices
T. Maho1, E. Robert1, M. Boudifa2, M.-P. Gellé3, F. Polidor4 and J.-M. Pouvesle1
1
GREMI-CNRS / Université d’Orléans, UMR7344, Orléans, France
² CRITT-MDTS, Charleville-Mézières, France
3
EA4691 BIOS, Reims, France
4
SOMINEX, Bayeux, France
Abstract: In the framework of the PLAS’STER project, a prototype based on a RF plasma
confined inside sterilization pouches for the treatment of medical devices has been
developed. In this work, optical emission spectroscopy was used to study the plasma
emission from VUV/UV to IR wavelengths range. The results showed the production of
strong UV radiation, radicals and atomic species depending on the process parameters.
Keywords: plasma medicine, medical devices sterilization, optical emission spectroscopy
1. Introduction
In France, 6.5 million surgeries are performed every
year. Each of these interventions presents the risk to
transmit an infectious agent to the patient by contact with
medical devices. These healthcare-associated infections
(HAI), named nosocomial infections, concern 7% of
hospitalized persons and are responsible of 4.000 deaths
[1].
Prolonged hospital stay, long-term disability,
massive additional financial burden, high costs for
patients and their family are some consequences of the
HAI. In developing countries, the risk is two to twenty
times higher and the proportion of infected frequently
exceeds 25%. In the world, 1.4 million persons suffer
from infectious complications acquired in hospital [2].
Sterilization of medical devices are crucial to world
public safety.
Conventional sterilization methods are based on moist
heat, ionizing irradiation and chemical treatment.
However, most recently developed materials are not
compatible with these standard sterilization treatments.
Because of the poor resistance of some sensitive materials
to sterilizing agents (moist heat, irradiations or chemical
treatments), new sterilization plasma based methods are
being developed. Allowing low processing temperature,
short treatment time and harmless operation for operators,
patients and materials, the plasma sterilization techniques
have been studied by several research groups. Low
pressure and low temperature plasmas have the potential
to fulfil all these requirements. Both glow discharge [3-7]
plasma and afterglow discharge [8, 9] plasma using
radio-frequency or microwave source allow a reduction of
bacterial population. In optimized conditions, the main
concern, is to maintain the sterile state following a 6-log
reduction. The identification and potential role of each
inactivation agent generated by the plasma are still under
study. Various gas mixtures could be used to optimize
the production of one of these agents and ensuring the
efficiency of the inactivation process.
Even if
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mechanisms of sterilization are not totally clarified,
several studies have shown that VUV/UV radiation can
induce strand breaks in DNA strands [10-12], reactive
species (O, OH, NO…) are known to be very efficient as
a biocidal medium and charge particles can lead to the
rupture of the outer membrane of bacterial cells [13].
In this work, the plasma chemistry with the optical
emission spectroscopy diagnostic (OES) has been
investigated. The aim was to identify the species created
to try to better understand the sterilization process under
our experimental conditions. In parallel, plasma
treatments on Pseudomonas aeruginosa have been done
to demonstrate the efficiency of our process for different
conditions.
2. Material and methods
2.1. Experimental setup
The schematic of the experimental set-up used for
diagnostics tests is presented in Fig. 1. It consists of a
cylindrical stainless-steel vacuum chamber (35 L)
equipped with suprasil windows. The plasma is generated
by a radio-frequency generator (13.56 MHz). Two coils
are connected in series to assist the discharge by a
magnetic field. The discharge chamber is pumped down
by a primary system and a turbo-molecular pump. Three
mass flow controllers are attached to the gas lines (Ar, O 2
and N 2 ).
1
Fig. 1. Schematic of the PLAS'STER reactor.
In contrast to all existing sterilization techniques using
vacuum or at atmospheric pressure plasmas [14], a
noteworthy key point in our process, is the sterility
conservation after sterilization treatment by the use of an
innovative technique. Covered by a patent (EP 2618851),
the medical device to be sterilized is placed in a special
sealed pouch allowing a gas flow. The pouch is made of
a medical grade of polyethylene (PE) provided by two
opercula made of Tyvek®. The pouches are set on the
powered electrode. By tuning the pressure difference
between the reactor chamber and the pouch, the plasma is
kept confined inside the pouches.
The major advantage of dealing with packaged medical
devices is to preserve their on-self sterility without any
additional precautions before further use.
2.2. Optical emission spectroscopy
The optical diagnostic was done by a combination of a
vacuum monochromator (ARC VM504) and a
photomultiplier tube (PMT). Emissions measurements in
VUV/UV region were performed with a Hamamatsu
R1080 PMT. Excitation spectra in VIS/IR were recorded
with a Hamamatsu R955 PMT. This device is also
equipped with motorized gratings controllable via
software.
Fig. 2. Photo showing the system allowing the VUV
spectrometry. Insert magnifies the connection to the
pouch.
The light emission from plasma is detected through a
cylindrical stainless-steel tube (length: 20 cm, diameter:
2.5 cm), which is connected to the pouch (see Fig. 2).
Two flange (DN40) were welded to the tube, one was
fixed to the reactor and the other one to the spectrometer.
A proof-connexion between this tube and the bag was
ensured by a gland. An MgF 2 window was used as the
vacuum window in the port between the vacuum chamber
and OES device. Thus, characterisation of VUV emission
was possible.
2.3. Preparation of bacteria suspensions
In accordance to the European standard EN1040,
Pseudomonas aeruginosa was chosen for the sterilization
process. Twenty microliters of 106 bacteria ml-1 was
spread on four sterile glass slide deposited on a Petri dish
used as carriers for the bacteria. The whole was placed
inside a sterilization pouch. The bacteria was exposed to
the plasma confined inside the pouch during the time
required to achieve at least 5log (EN1040).
After treatment, the number of colonies of the bacteria
was immediately counted.
Then, the survivability
profiles, which reflected the number of microorganisms
that remained alive versus the sterilization time was
plotted. Each point is the mean value of 9 experiments.
3. Results
3.1. VUV/UV spectrum
VUV/UV light emission spectra from oxygen plasma
for two different RF powers are shown in Fig. 3. The
radiation are originating from N 2 *(a-X), CO*(A-X) below
220 nm, λ = 200-280 from NO*(A-X, C-X) and above
280 nm from OH*(A-X). Atomics lines from C (156,
163, 165.7 and 193.1 nm) and N (174.5 nm) were also
identified (see Table 1).
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Fig. 3. VUV/UV spectrum of oxygen plasma from 100 to
300 nm.
Table 1. Identification of the spectral emission from 100
to 300 nm in oxygen plasma at 50 W.
Species
NO γ
NO δ
N 2 (a-X)
CO(A-X)
C
N
OH(A-X)
Wavelengths (nm)
215.5, 226.2, 230.9, 237, 244.7 247.8,
255, 259.5, 267.1
198.5, 206.1
150.8, 153.0
140.9, 143.5, 146.3, 149.4, 155.9,
157.7, 159.7, 171.2, 172.9, 179.3,
181.1, 183, 186, 187.9, 189.8, 195,
197, 201.3, 202.7, 204.6, 208.9, 211.3,
219.7
156, 163, 165.7, 193.1
174.5
282.9, 288.2, 289.3, 296.2
We discussed previously that one of the sterilization
mechanism could be the DNA destruction by VUV/UV
photons. Thus, it would be interesting to increase the UV
radiation to improve the process efficiency in the
inactivation of micro-organisms. In this regard, the RF
power was increased up to 150 W, where the VUV/UV
radiation intensity was multiplied by 3 below 220 nm, by
2.5 between 220 and 250 nm and by 2 above 250 nm.
The formation of carbon monoxide is probably due to
the slight etching of the wall pouch. X-ray photoelectron
spectroscopy (XPS) analysis has been performed to detect
chemical changes at the surface before and after
treatment. The measurements showed some grafting of
C-O and C=O functionalities. Otherwise, FTIR analysis
of the pouch showed no change in the macromolecular
structure of the PE which assumes that the barrier
properties of the sterilizing pouch are not affected with
the low RF power plasma. However, in an optimization
approach of the whole process, the increase in the RF
power would likely be limited to a certain critical point,
since the integrity of the pouch and the medical devices is
required.
So far, all the biological tests conducting to a 6-log
reduction of Pseudomonas aeruginosa in 45 min have
been done at 25 W. The first step in the process
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optimization should be the definition of a higher power
effect.
The optical measurements in argon and nitrogen plasma
also showed emissions of N 2 *(a-X, C-B), CO*(A-X),
NO*(A-X, C-X) and OH*(A-X). The difference between
the three gases has been the reduction of CO* and the
excitation of NO and OH. According to Fig. 4, several
bands of NO δ and NO γ are more intense in argon plasma
than in nitrogen and oxygen plasma. Therefore, the
hydroxyl radical would be promoted in an oxygen plasma.
One band in nitrogen, 2 bands in argon and 4 bands in
oxygen are clearly identified from the OH(A-X)
molecular system.
Furthermore, because of the pouch etching in oxygen
plasma, argon could be a good alternative to study the
VUV/UV radiation effect on bacteria.
3.2. VIS/IR spectrum
The VIS/IR wavelengths were scanned using the PMT
R955. The obtained spectrum when feeding the reactor
with only oxygen is shown in Fig. 5. The identified
species are summarized in Table 2. The typical oxygen
atoms emissions lines were observed at 777 and 844 nm.
Other lines come from impurities or from products of the
oxygen plasma with the surrounding pouch. Five intense
bands of CO*(B-A) were identified at 451.1, 483.5, 519.8,
561 and 607.9 nm. The hydrogen peak Hα at 656 nm
may be due to the reaction of oxygen plasma with pouch
walls. The second and the first positive systems of
nitrogen were represented, respectively, from 300 to 435
nm and from 575 to 775 nm. The presence of N 2 +* at
391.4, 427.8 and 470.9 nm give an indication of the
energy quantity released, since the required excitation is
18.6 eV.
3
Table 2. Identification of the spectral emission from 300
to 900 nm in oxygen plasma at 50 W.
Species
N 2 (C-B)
N 2 (B-A)
N2+
CO(A-X)
OH(A-X)
O
H
Wavelengths (nm)
313.6, 315.9, 337.1, 353.6, 357.7, 371,
380.4, 394.3, 420, 434.3
580.4, 590.6, 595.9, 601.3, 606.9,
639.5, 687.5, 727.3, 738.6, 750.4,
789. 6, 804.7, 872.2
391.4, 427.8, 470.9
451.1, 483.5, 519.8, 561, 607.9
306.7, 308.9
777.1, 844.6
656.6
Nitrogen plasma was dominated by emissions of the
first positive system below 450 nm and of the second
positive system above 600 nm. The N 2 +* ion has also
been identified at 391.4, 427.8 and 470.9 nm.
Argon plasma was characterized by several typical
emission lines from 450 to 900 nm. OH*(A-X) was
identified between 306 and 310 nm and the most intense
bands of N 2 *(C-B) were observed below 340 nm.
3.3. Biological tests
All the biological tests are done in EA4691 BIOS
laboratory. The reactor geometry is slightly different but
the parameters are the same (RF power, gases, coil and
pressure).
The microbiological results for the treatment of
Pseudomonas aeruginosa are shown in Fig. 6. Each data
point is the mean value of nine samples. First, we have
observed that the vacuum had a slight effect on the
bacteria with a 1.55log reduction in 45 minutes. Then,
the used of an oxygen, nitrogen or argon based plasma
have allowed a 6log reduction in 45 minutes.
Thermographic strips have demonstrated that the
temperature process did not exceed 40 °C which is not
enough to kill bacteria.
Fig. 4. Comparison of NO* and OH* emission from 180
to 300 nm in oxygen, nitrogen and argon plasma at 50 W.
Fig. 6. Survival curve for Pseudomonas aeruginosa
exposed to the plasma at 25 W. Gas flow is 0.4 sccm.
Fig. 5. VIS/IR spectrum of oxygen plasma from 300 to
900 nm at 50 W.
4
The survival curve shows a two-step kinetics,
consisting of a fast initial reduction by 4 log, followed by
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a slow reduction down to 1 log. There are more
pronounced in oxygen and nitrogen based plasma. It is
highly probable that the treatment time can be reduced by
rising the power up to 150 W.
4. Conclusion and discussion
A preliminary study of low pressure low temperature
plasma confined in sterilization pouches has been carried
out. Optical Emission Spectroscopy analysis has been
essentially presented in this work, giving constructive
information’s on the plasma chemistry.
Both UV
radiations and reactive species (OH, NO, O) may play a
role in the sterilization process documenting in Fig. 6.
The effectiveness of the method has been demonstrated
for sterilization. The control of the reactive species has
been made possible by tuning the gas nature which
mainly depends on parameters. For example, to estimate
the role of UV radiation without etching the pouch, it
would be interesting to make further bacteriologic tests
with argon plasma, instead of oxygen, with higher levels
of power.
The chemical measurements have shown that pure
oxygen plasma can induced surface modifications on
pouches walls without altering barrier properties. Plasma
parameters should be chosen in such a way to found a
compromise between plasma efficiency and damage of
the sterilizing pouches.
Biological tests have been performed on Pseudomonas
aeruginosa, the standardized 6-log reduction was obtained
with different gas in 45 min at 25 W. Undergoing
bacteriological tests combined to plasma diagnostics
should lead to a better understanding of the sterilization
mechanisms.
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5. Acknowledgements
This work is supported by the ANR-12-TECS-0007
project PLAS’STER.
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